Figure 2. RB1 Loss Is Associated With Azenosertib Sensitivity in TP53‑Mutant Cancer Cell Lines 0.001 0.01 0.1 1 10 100 -1.0 -0.5 0.0 0.5 1.0 1.5 Concentration (µM) G R V al ue 0.001 0.01 0.1 1 10 100 -1.0 -0.5 0.0 0.5 1.0 1.5 Concentration (µM) G R V al ue DMS53 TP53 mut/ RB1 wt TP53 mut/ RB1 mut SW1271 NCI-H1048 NCI-H1963 NCI-H446 NCI-H510 NCI-H524 NCI-H209 NCI-H526 MDA-MB-231 MDA-MB-468 BT549 TP53 mut/ RB1 wt TP53 mut/ RB1 mut A B Small cell lung cancer (SCLC) Triple-negative breast cancer (TNBC) (A) Small cell lung cancer and (B) triple‑negative breast cancer cells were seeded in 96‑well plates and treated with azenosertib for 72 hours. Cell viability was measured using the CellTiter‑Glo assay to calculate GR inhibition values. GR and GRmax were determined using pre‑ and post‑treatment cell viability and the GR calculator.8 GR > 0: growth inhibitory effect; GR = 0: cytostatic effect; GR < 0: cytotoxic effect; GR = ‑1: complete cytotoxicity. Figure 3. RB1 Knockdown Sensitizes Cancer Cells to Azenosertib and Results in Higher Levels of DNA Damage Upon Azenosertib Treatment DMS53 (SCLC) TP53 mut, RB1 WT MDA-MB-231 (TNBC) TP53 mut, RB1 WT MDA-MB-231 (TNBC) TP53 mut, RB1 WT 0.0001 0.001 0.01 0.1 1 10 100 -1.0 -0.5 0.0 0.5 1.0 1.5 G R V al ue 0.0001 0.001 0.01 0.1 1 10 100 -1.0 -0.5 0.0 0.5 1.0 1.5 Concentration (µM) Concentration (µM) G R V al ue 0 48 144 192 +443% +344% +2% +373% +33% -47% 0 2 4 6 96 Elapsed Time (hr) shCtrl shRB1 Ra ti o of C on flu en ce (N or m al is ed to tr ea tm en t s ta rt ) 0 48 144 192 0 2 4 6 96 Elapsed Time (hr) Ra ti o of C on flu en ce (N or m al is ed to tr ea tm en t s ta rt ) 0 0.1 0.3 1 Azenosertib (µM) RB1 β-Actin p-CDK1 (Y15) γH2AX (S139) p-CHK1 (S345) cl-Caspase 7 cl-Caspase 3 β-Actin Ta rg et en ga ge m en t shCtrl 0 0.1 0.3 1 shRB1 0Azenosertib (μM) Azenosertib (μM) 0.1 0.3 1 shCtrl 0 0.1 0.3 1 shRB1 D N A da m ag e A po pt os is p-CHK1 (S345) γH2AX (S139) cl-Caspase 3 cl-Caspase 7 0 0.1 0.3 1 Azenosertib (µM) RB1 β-Actin p-CDK1 (Y15) γH2AX (S139) p-CHK1 (S345) cl-Caspase 7 cl-Caspase 3 β-Actin Ta rg et en ga ge m en t shCtrl 0 0.1 0.3 1 shRB1 0 0.1 0.3 1 shCtrl 0 0.1 0.3 1 shRB1 D N A da m ag e A po pt os is p-CHK1 (S345) γH2AX (S139) cl-Caspase 3 cl-Caspase 7 1.0 2.1 4.5 6.3 1.2 3.2 9.0 13.2 1.0 1.2 2.5 8.1 1.0 1.1 3.2 12.3 1.0 1.0 1.4 1.0 1.9 0.9 1.1 7.0 1.0 1.1 1.6 3.7 1.1 0.7 2.5 6.5 1.0 1.1 1.9 4.8 1.4 1.2 2.9 8.0 1.0 1.1 1.5 5.6 1.1 1.3 6.6 18.0 1.0 1.0 1.3 1.3 2.4 2.4 6.1 10.9 1.0 1.2 1.2 3.1 2.5 3.0 3.4 10.0 shCtrl shRB1 shCtrl shRB1 DMSO Azenosertib 0.37 µM Azenosertib 1.11 µM Treatment Start Fold change relative to control 0246 Fold change relative to control 0246 A B C D G E F (A) DMS53 RB1 knockdown isogenic cells were treated with azenosertib for 72 hours to assess GR values. (B‑C) Cells were treated with azenosertib (0, 0.1, 0.3, and 1 µM) for 16 hours, and protein expression of the indicated markers was analyzed using traditional Western blot and JESS automated Western platform. (D‑F) MDA‑MB‑231 RB1 knockdown isogenic cells were similarly treated, and protein expression was analyzed. (G) MDA‑MB‑231 RB1 knockdown isogenic cells were treated with azenosertib (0, 0.37, 1.11 µM) for 8 days, and proliferation and confluence were measured using Incucyte. Figure 4. RB1 Inducible Overexpression Desensitizes Cancer Cells to Azenosertib NCI-H1048 (SCLC) TP53 mut, RB1 mut MDA-MB-468 (TNBC) TP53 mut, RB1 mut 0.0001 0.001 0.01 0.1 1 10 100 -1.0 -0.5 0.0 0.5 1.0 1.5 Concentration (µM) G R V al ue 0.0001 0.001 0.01 0.1 1 10 100 -1.0 -0.5 0.0 0.5 1.0 1.5 Concentration (µM) G R V al ue Azenosertib (μM) Azenosertib (μM) 0 0.3 1 Dox (µM) Azenosertib (µM) RB1 β-Actin p-CDK1 (Y15) γH2AX (S139) p-CHK1 (S345) cl-Caspase 7 cl-Caspase 3 β-Actin Ta rg et en ga ge m en t RB1 IOE 0 0.3 1 0 0.3 1 0 0.3 - 0.05 0.1 0.5 1 D N A da m ag e A po pt os is 0 0.3 1 w/o Dox 0 0.3 1 Dox 0.05 µM 0 0.3 1 Dox 0.1 µM 0 0.3 1 Dox 0.5 µM p-CHK1 (S345) γH2AX (S139) 0 0.3 1 Dox (µM) Azenosertib (µM) RB1 β-Actin p-CDK1 (Y15) γH2AX (S139) p-CHK1 (S345) cl-Caspase 7 cl-Caspase 3 β-Actin Ta rg et en ga ge m en t RB1 IOE 0 0.3 1 0 0.3 1 0 0.3 - 0.05 0.1 0.5 1 D N A da m ag e A po pt os is 0 0.3 1 w/o Dox 0 0.3 1 Dox 0.05 µM 0 0.3 1 Dox 0.1 µM 0 0.3 1 Dox 0.5 µM p-CHK1 (S345) γH2AX (S139) cl-Caspase 3 cl-Caspase 7 cl-Caspase 3 cl-Caspase 7 1.0 3.5 13.0 0.9 1.7 5.9 0.9 1.4 4.0 0.8 1.0 1.5 1.0 1.1 10.5 1.0 1.0 4.7 1.0 1.0 3.0 1.0 1.0 1.5 1.0 1.2 8.7 0.8 1.0 4.8 0.7 0.9 2.3 0.6 0.7 1.1 1.0 1.3 9.5 1.1 1.2 6.5 0.9 1.2 3.2 0.8 0.9 1.7 1.0 3.110.4 0.8 1.9 9.8 0.8 1.5 6.0 0.7 0.9 1.6 1.0 0.8 7.7 1.0 1.1 1.7 0.8 1.4 2.5 0.7 0.9 0.7 1.0 2.9 8.7 0.5 1.3 2.0 0.3 1.2 2.3 0.5 0.5 1.7 1.0 1.2 3.1 1.3 1.2 2.4 0.7 0.9 2.1 0.9 0.7 1.2 Ctrl OE RB1 IOE RB1 IOE + Dox 0.1 µM Ctrl OE RB1 IOE RB1 IOE + Dox 0.5 µM Fold change relative to control 0246810 Fold change relative to control 02468 A B C D E F (A) NCI‑H1048 RB1 overexpression isogenic cells were treated with Dox for 24 hours prior to azenosertib treatment for 72 hours, and GR values were assessed. (B‑C) Cells were treated with azenosertib (0, 0.3 and 1 µM) for 16 hours, and protein expression was analyzed via Western blot and JESS. (D‑F) MDA‑MB‑468 RB1 overexpression isogenic cells were treated under the same conditions and analyzed accordingly. Figure 5. Azenosertib Promotes Higher G1/S Transition and More DNA Damage in RB1‑Knockdown TNBC Cells In Vitro M D A -M B- 23 1 sh Ct rl M D A -M B- 23 1 sh RB 1 M D A -M B- 23 1 sh Ct rl Cell cycle phase M D A -M B- 23 1 sh RB 1 M D A -M B- 23 1 sh Ct rl DNA damage M D A -M B- 23 1 sh RB 1 DNA content DMSO 0.04% 0.55% 7.66% 0.03% 1.72% 10.9% Azeno 0.3 µM Azeno 1 µM DMSO Ed U Ed U Azeno 0.3 µM Azeno 1 µM DNA content DMSO Azeno 0.3 µM Azeno 1 µM DMSO Azeno 0.3 µM Azeno 1 µM 0 1 µM 0.3 µM DMSO 20 40 60 % of Cells 80 100 0 1 µM 0.3 µM DMSO 20 40 60 % of Cells 80 100 0 1 µM 0.3 µM DMSO % of γH2AX+ Cells 105 1050 1 µM 0.3 µM DMSO % of γH2AX+ Cells Subset Name γH2AX+ Cell Cycle Subset Name γH2AX+ Cell Cycle Sub G1 G1 EdU+ S EdU- S G2/M Sub G1 G1 EdU+ S EdU- S G2/M Sub G1 G1 EdU+ S EdU- S G2/M Sub G1 G1 EdU+ S EdU- S G2/M A B C (A‑C) MDA‑MB‑231 RB1 knockdown isogenic cells were treated with azenosertib (0, 0.3 and 1 µM) for 24 hours. Cell cycle distribution was analyzed by flow cytometry using the indicated markers, quantified with FlowJo, and plotted using GraphPad Prism. The percentage of γH2AX+ cells is highlighted in red in (A). Figure 6. Azenosertib Demonstrated Greater Anti‑Tumor Activity in RB1‑Mutant CDX Models 0 5 10 15 20 25 30 -20 -10 0 10 20 M ea n Δ BW (% ) 0 5 10 15 20 25 30 -20 -10 0 10 20 M ea n Δ BW (% ) 0 10 20 30 -20 -10 0 10 20 M ea n Δ BW (% ) 0 10 20 30 40 50 60 -20 -10 0 10 20 M ea n Δ BW (% ) 0 5 10 15 20 25 30 0 600 1200 1800 2400 Days post-treatment 49% TGI M ea n TV ± S EM (m m 3 ) 0 5 10 15 20 25 30 0 500 1000 1500 2000 Days post-treatment M ea n TV ± S EM (m m 3 ) 90% TGI 0 10 20 30 0 600 1200 1800 Days post-treatment M ea n TV ± S EM (m m 3 ) 50% TGI 0 10 20 30 40 50 60 0 600 1200 1800 Days post-treatment M ea n TV ± S EM (m m 3 ) 87% TGI Vehicle QD Azenosertib 80 mg/kg QD Vehicle QD Azenosertib 80 mg/kg QD Vehicle QD Azenosertib 80 mg/kg QD Vehicle QD Azenosertib 80 mg/kg QD DMS53 (SCLC) TP53 mut, RB1 WT NCI-H146 (SCLC) TP53 mut, RB1 mut MDA-MB-231 (TNBC) TP53 mut, RB1 WT MDA-MB-468 (TNBC) TP53 mut, RB1 mut A B C D NOD/SCID mice were subcutaneously inoculated with DMS53 (A), NCI‑H146 (B), MDA‑MB‑231 (C), and MDA‑MB‑468 (D) cells. Treatment was initiated when mean tumor volume reached 200 mm³ (n=8/group for SCLC, n=10/group for TNBC). The inset graph depicts the mean ΔBW. All treatments were well tolerated (ΔBW ≤15%). TGI was calculated as: TGI = (1 ‑ [Td ‑ T0] / [Cd ‑ C0]) × 100%. RESULTSBACKGROUND • Azenosertib is a potent and selective WEE1 inhibitor currently in clinical development. WEE1 regulates the G1/S and G2/M cell cycle checkpoints by inhibiting CDK1 and CDK2, preventing cells with damaged DNA from progressing through the cell cycle • Inhibition of WEE1 by azenosertib accelerates cell cycle progression, leading to increased replication stress, premature mitotic entry, and DNA damage accumulation, ultimately resulting in mitotic catastrophe and cancer cell death1 • RB1 is a crucial tumor suppressor that regulates the G1/S cell cycle transition by negatively regulating E2F transcription factors. Loss of RB1 leads to uncontrolled cell cycle progression from G1 to S phase and increased replication stress, especially in TP53 mut cancer cells which already have G1/S defect2‑4 • RB1 loss is highly prevalent in aggressive cancers such as SCLC and TNBC, where it correlates with poor prognosis and therapy resistance.5‑7 Both tumor types are also enriched with TP53 mut • Given that combined loss of TP53 and RB1 promotes G1/S checkpoint defect and replication stress, we hypothesized that these cells would be highly sensitive to WEE1 inhibition by azenosertib Figure 1. Mechanism of Action of Azenosertib RB1 Azenosertib WEE1 RB1 E2F inactive E2F Cyclin CDK2 Cyclin CDK1 DNA damage CDK2 active CDK1 Active Mitotic catastrophe and death G2 M G1 S G1/S Checkpoint G2/M Checkpoint DNA damage accumulates DNA damage Azenosertib WEE1 Azenosertib WEE1 Azenosertib WEE1 E2F active E2F Cyclin CDK2 Cyclin CDK1 CDK2 active CDK1 Active Increased mitotic catastrophe and death G2 M G1 S G1/S Checkpoint G2/M Checkpoint DNA damage accumulates The Effect of Azenosertib on Cancer Cells The Effect of Azenosertib on RB1-Deficient Cancer Cells CONCLUSIONS • RB1 loss of function is associated with azenosertib sensitivity in TP53‑mutant SCLC and TNBC cell lines, as demonstrated by greater growth inhibition after azenosertib treatment • RB1 knockdown sensitizes RB1 WT cancer cells to azenosertib, while RB1 overexpression desensitizes RB1‑deficient cells to azenosertib treatment in vitro • Mechanistic analysis shows that azenosertib treatment induces more G1/S cell cycle transition and higher levels of DNA damage and apoptosis in RB1‑knockdown cells • In vivo studies revealed that azenosertib induces greater TGI in RB1‑deficient TP53‑mutant models compared to RB1 WT TP53‑mutant models • These findings suggest that RB1 loss is associated with sensitivity to WEE1 inhibition by azenosertib in preclinical models, and patients whose tumors harbor both RB1 loss and TP53 mutation may be more likely to benefit from azenosertib therapy References 1. di Rorà AGL, et al. J Hematol Oncol. 2020;13(1):126. 2. Adon T, et al. RSC Adv. 2021;11(47):29227‑29246. 3. Huang MF, et al. Cancers (Basel). 2024;16(8):1558. 4. Engeland K, et al. Cell Death Differ. 2022;29(5):946‑960. 5. Jones RA, et al. J Clin Invest. 2016;126(10):3739‑3757. 6. Robinson TJ, et al. PLoS One. 2013;8(11):e78641. 7. Mandigo AC, et al. Clin Cancer Res. 2022;28(2):255‑264. 8. Clark NA, et al. BMC Cancer. 2017;17(1):698. Acknowledgments This study was sponsored by Zentalis Pharmaceuticals, Inc. Animal studies were performed at Pharmaron, Beijing. Editorial support for this poster was provided by Second City Science, LLC. Additional Information For more information on this study, visit www.zentalis.com or contact gkim @ zentalis . com. Abbreviations Azeno, azenosertib; ΔBW, percent change in body weight; CDK, cyclin dependent kinase; Chk1, checkpoint kinase; CDX, cell line‑derived xenograft; cl‑cleaved caspase; Ctrl, control; Dox, doxycycline; DMSO, dimethyl sulfoxide; EdU, 5‑ethynyl‑2′‑deoxyuridine; G1/S, GAP1/ synthesis; G2/M, GAP2/mitosis; γH2AX, phospho‑histone H2AX; GR, growth rate; IOE, inducible overexpression; JESS, Jess Automated Western Blot System; mut, mutated; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; p‑CDK1, phosphorylated cyclin dependent kinase 1; p‑CHK1, phospho‑checkpoint kinase 1; QD, daily; SCLC, small cell lung cancer; SEM, standard error of mean; sh, short hairpin; TGI, tumor growth inhibition; TNBC, triple‑negative breast cancer; TP53, tumor protein p53; TV, tumor volume; WT, wildtype. PRESENTED AT: American Association for Cancer Research (AACR), April 25‑30, 2025, Chicago, Illinois, USA Poster #372 Loss of RB1 Sensitizes TP53‑Mutated Cancer Cells to WEE1 Inhibition by Azenosertib Daehwan Kim, Olivier Harismendy, Catherine Lee, Erika Cabrera, Harshit Shah, Danielle Jandial, Doris Kim, Mark R. Lackner, Jianhui Ma Zentalis Pharmaceuticals, Inc., San Diego, CA, USA. Exhibit 99.1

Figure 3. RECIST Responders Showed a Higher Molecular Response Rate P = 0.012 Odds ratio (95% CI) = 0.26 (0.090-0.75) MR+ (N) MR- (N) RECIST Responder 20 5 RECIST Non-responder 50 48 PPV (95% CI) 28% (19-40%) NPV (95% CI) 91% (80-96%) 20/25 (80% MRR) cOR 40/71 (56% MRR) SD 9/26 (35% MRR) 80% of responders per RECIST had a molecular response 91% of patients without molecular response were non-responders per RECIST PD 29% 57% 13% 1% 9% 58% 32% mPD mSD mPR mCR cOR Best Overall Response (RECIST) SD PD NA C1D1 C2D1 C3D1 C1D1 C2D1 C3D1 C1D1 C2D1 C3D1 −100 −50 0 50 100 Visit Pe rc en t c ha ng e in T P5 3 V A F fr om b as el in e (% ) 0 25 50 75 100 Fr ac ti on o f p ati en ts (% ) p = 0.0094 MR+ (N=70) MR− (N=53) A B A. MRR is highest in CR/PR RECIST response and lowest in PD. Dashed lines indicate the staged MR cutoffs. The color of lines indicate molecular response stages defined in Figure 2. B. Clinical response was enriched in MR+ cohort. Statistical significance was calculated using Chi‑squared test and Fisher’s exact test (two‑sided), respectively. Figure 4. Molecular Responders Showed Greater Tumor Size Reduction MR stage mCR mPR mSD mPD Be st p er ce nt c ha ng e fr om b as el in e in s um o f d ia m et er (% ) −100 −30 0 20 >100 Complete molecular responders showed a median target lesion size reduction of 31% based on RECIST criteria MR stage mCR mPR mSD mPD N 26 42 32 20 P-value 5.1e-5 Median tumor size change -30.6% -18.2% -7.3% 5.7% Best percent change in the sum of the diameters of target lesions is shown with color indication of molecular response stage. The inset table shows the median change across MR stages. Statistical significance from Kruskal‑Wallis test. Dashed lines indicate 20% and ‑30% RECIST criteria cutoffs. Figure 5. Molecular Responders Showed a Longer Time‑To‑Progression 70 55 39 20 8 1 0 0 0 53 31 15 7 3 1 1 1 1MR− MR+ Patients at risk 38 35 25 14 6 1 0 0 0 31 27 13 6 3 1 1 1 1MR− MR+ Patients at risk 0 0.25 0.5 0.75 1.0 0 0.25 0.5 0.75 1.0 0 2 4 6 8 10 12 14 16 Months 0 2 4 6 8 10 12 14 16 Months Entire cohort (N=123) Patients with stable disease at the first assessment (SD1A; N=69) Su rv iv al p ro ba bi lit y Su rv iv al p ro ba bi lit y MR+ MR− MR+ MR− MR+ MR- mTTP (months) (95% CI) 5.49 (4.14-6.70) P-valueHR (95% CI) 0.43 (0.29-0.65) 3.8e-5 2.69 (2.56-3.84) MR+ MR- mTTP (months) (95% CI) P-valueHR (95% CI) 6.14 (4.73-8.54) 0.44 (0.25-0.78) 3.8e-3 3.56 (2.73-4.37) TTP is compared between MR+ and MR‑ groups (left) in the entire evaluable cohort (N=123) and (right) in the patients whose first overall response was stable disease (N=69). mTTP and HR with 95% CI are shown in inset tables. P‑values are from log‑ranked test. Figure 6. Complete and Confirmed Molecular Responses Further Stratified Time‑to‑Progression mPR mCR mSD mPD Patients at risk 28 23 19 10 5 0 0 0 0 42 32 20 10 3 1 0 0 0 33 21 11 5 2 1 1 1 1 20 10 4 2 1 0 0 0 0 uMR+ cMR+ cMR− Patients at risk 38 37 29 15 6 1 0 0 0 10 8 3 2 0 0 0 0 0 17 15 6 5 3 1 1 1 1 Entire cohort (N=123) Patients with 2 on-treatment timepoints (N=65) Patients with 2 on-treatment timepoints (N=65) 0 0.25 0.5 0.75 1.0 0 0.25 0.5 0.75 1.0 0 2 4 6 8 10 12 14 16 Months Su rv iv al p ro ba bi lit y 0 2 4 6 8 10 12 14 16 Months Su rv iv al p ro ba bi lit y mPDmSDmPRmCR cMR−uMR+cMR+ mCR mPR mTTP (months) (95% CI) 6.47 (5.42-NA) 4.14 (3.25-6.14) mSD mPD 2.73 (2.56-4.14) 2.69 (1.41-4.11) P-value mCR vs mSD/mPD 2.2e-5 HR (95% CI) mCR vs mSD/mPD 0.30 (0.17-0.54) mCR mPR Confirmed MR+ 16 22 mSD mPD 0 0 3 4 3 0 0 0 10 7 cMR+ uMR+ mTTP (months) (95% CI) 6.14 (5.39-8.48) 3.84 (3.25-NA) cMR- 2.76 (2.66-7.72) P-value cMR+ vs cMR- 0.014 HR (95% CI) cMR+ vs cMR- 0.46 (0.24-0.87) Unconfirmed MR+ Confirmed MR- Median TTP was 6.14 months for confirmed MR compared with 2.8 months for cMR- Complete molecular response (mCR) and confirmed molecular response appeared independent Median TTP for mCR was 6.5 months, compared with 2.7 months in mPD TTP is illustrated (left) across staged MR groups in the entire evaluable cohort (N=123) and (right) across confirmed MR groups in the patients whose C2D1 and C3D1 samples are both available (N=65). Confirmed MR+ (cMR+) is defined as MR+ at both C2D1 and C3D1 timepoints, unconfirmed MR+ (uMR+) as MR+ at either timepoint, and confirmed MR‑ (cMR‑) as MR‑ at both timepoints. mTTP and HR with 95% CI are shown in inset tables. P‑values are from log‑ranked test. Figure 7. Molecular Response Preceded RECIST Outcome in Patients with Stable Disease at the First Assessment 0 6 12 18 24 30 36 42 48 54 60 66 72 Weeks from C1D1 Patients with stable disease at the first assessment (SD1A; N=69) Group 1 Group 2 Group 3 Group 4 Group 5 RECIST response Molecular response Response Stable disease Not evaluable Progression mCR/mPR mSD mPD Group Number of patients MR BOR MR-BOR correlation Lead time to BOR (weeks) Group 1 5 MR+ cOR Yes 14 (12-19) Group 2 2 MR- cOR No - Group 3 19 MR+ SD No - Group 4 26 MR- SD Yes 8 (2-31) Group 5 17 MR+/MR- NAa - - • MR+ identified 14 weeks prior to response (Group 1; n=5) • MR- identified 8 weeks prior to progression (Group 4; n=26) Timepoints of MR and RECIST overall response calls are shown for the SD1A cohort (N=69) (Figure on left). Patients were classified into 5 groups based on MR and BOR (Table on Right). For the groups in which MR predicted clinical response, median time benefit was calculated. aNot available due to treatment ongoing or discontinued without progression. RESULTSBACKGROUND • Azenosertib is a novel selective WEE1 kinase inhibitor showing promising activity in the treatment of gynecologic tumors, including HGSOC1,2 • The clinical development of azenosertib in HGSOC is conducted via several efficacy studies using objective response rate and progression‑free survival as primary and secondary endpoints • Molecular response (MR), as measured by longitudinal changes of tumor DNA fraction in cell‑free DNA (cfDNA), can represent a cost‑effective, and early efficacy endpoint, which needs further evaluation in HGSOC clinical studies METHODS Figure 1. (A) Clinical Setting for MR Analysis in Azenosertib‑Treated HGSOC. (B) Description of Azenosertib‑Treated HGSOC Cohort for MR Analysis Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 ••• 0Time (week) HGSOC 3 6 Plasma cfDNA (TP53 VAF) Azenosertib treatment Radiological tumor assessment (RECIST v1.1) 9 12 HGSOC patients treated with ≥300 mg of azenosertib (QD) on 5:2 intermittent dosing (N=220) Excluded (N=64) • Plasma not collected (N=55) • cfDNA NGS assay pending (N=9) Excluded (N=17) • cfDNA NGS assay QC failed Excluded (N=16) • No baseline TP53 variant detected Plasma cfDNA NGS assay processed (N=156) Eligible for MR analysis (N=139) Evaluable for MR analysis (N=123) A B A. Retrospective MR study was designed with HGSOC patients enrolled in azenosertib studies, ZN‑c3‑001 (NCT041583363), ZN‑c3‑005/DENALI (NCT051288254) and ZN‑c3‑006/MAMMOTH (NCT051988045). Patients treated with monotherapy at a total daily dose of 300 mg or higher of azenosertib (QD) on a 5:2 intermittent dosing schedule were selected. Longitudinal cfDNA collection was implemented in ZN‑c3‑001 after a protocol amendment in 2023 so only includes a subset of patients enrolled in this trial. Plasma samples were collected every 3 weeks and analyzed by Tempus xF+ Liquid Biopsy Assay to calculate TP53 variant allele fraction in cfDNA. Radiological tumor assessment (RECIST v1.1) was conducted every 6 weeks. Efficacy was extracted from an unlocked clinical database (data cutoff: 1/27/2025). B. All patients were treated with azenosertib monotherapy at a total daily dose of 300 mg or higher (QD) on a 5:2 intermittent dosing schedule. Among 139 patients whose plasma samples were properly collected and successfully analyzed, 123 patients showed TP53 variants detectable at baseline and were considered the evaluable cohort. On‑treatment plasma samples were collected at C2D1 and/or C3D1 to evaluate molecular response on treatment. Clinical response endpoints including best overall response, best tumor size reduction and time‑to‑progression (TTP) were used to validate molecular response. acOR defined as confirmed response per RECIST 1.1 criteria in subjects with at least one post‑baseline assessment. Figure 2. TP53 VAF Change Distribution and MR Definition MR stage Definition Molecular complete response (mCR) mVAF change = -100% Molecular partial response (mPR) -100% < mVAF change < -50% Molecular stable disease (mSD) -50% ≤ mVAF change ≤ 0% Molecular progressive disease (mPD) 0% < mVAF change MR stage mCR mPR mSD mPD 0 10 20 30 40 −100 −50 0 50 >100 Percent change in TP53 VAF from baseline (%)a N um be r of p ati en ts mCR mPR MR+ MR- mSD mPD Distribution of percent change in TP53 variant allele fraction at the earliest available timepoint (either C2D1 or C3D1) is shown with the ranges of different MR stages (left). The definition of different MR stages are described in the table (right). MR positivity includes mCR and mPR, whereas MR negativity includes mSD and mPD. aMedian VAF used in multiple TP53 variants detected. CONCLUSIONS • The study presents the largest evaluation of cfDNA‑based MR in HGSOC treated with an investigational drug (N=123 patients) • Clinical validity: cfDNA TP53‑based MR showed significant associations with BOR, RECIST tumor size change, and TTP – In particular, complete reduction or clearance of TP53 VAF identified patients with the greatest reduction in tumor size and longest TTP • Clinical utility: MR may predict outcome in HGSOC patients with stable disease at first RECIST assessment – Molecular responders had longer TTP – MR established within the first or second cycle of treatment provided an early indication of RECIST response (median 14 weeks) or progression (median 8 weeks) • Response characterization: Complete MR and confirmed MR appeared independent References 1. Pasic A, et al. Presented at the AACR 2022 Annual Meeting. Abstract number CT148. 2. Liu J, et al. Presented at the ASCO 2023 Annual Meeting. Abstract number GOG‑3072. 3. https://clinicaltrials.gov/study/NCT04158336. 4. https://clinicaltrials.gov/study/NCT05128825. 5. https://clinicaltrials.gov/study/NCT05198804. Acknowledgments This study is sponsored by Zentalis Pharmaceuticals, Inc. Editorial support for this poster was provided by Second City Science, LLC. Additional Information For more information on this study, visit www . zentalis . com or contact jjeong @ zentalis . com. Abbreviations 5:2, 5 days on, 2 days off; BOR, best overall response; CD, cycle day; cfDNA, cell‑free DNA; CI, confidence interval; cMR, confirmed molecular response; cMR+, confirmed molecular response positivity; cMR‑, confirmed molecular response negativity; cOR, confirmed objective response; HGSOC, high‑grade serous ovarian cancer; HR, hazard ratio; mCR, molecular compete response; mPD, molecular progressive disease; mPR, molecular partial response; MR, molecular response; mSD, molecular stable disease; mTTP, median time‑to‑progression; mVAF, median variant allele fraction; NA, not assessable; NE, not evaluable; NGS, next generation sequencing; NPV, negative predictive value; ORR, objective response rate; PD, progressive disease; PPV, positive predictive value; QC, quality control; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; SD1A, stable disease at first assessment; TTP, time‑to‑progression; uMR+, unconfirmed molecular response positivity; VAF, variant allele fraction.PRESENTED AT: American Association for Cancer Research (AACR), April 25‑30, 2025, Chicago, Illinois, USA Poster #3254 Cell‑Free DNA Molecular Response Predicts Clinical Efficacy in HGSOC Patients Treated With Azenosertib Jinkil Jeong1, Jianhui Ma1, Monah Abed1, Heekyung Chung1, Doris Kim1, Nandini Molden1, Divya Rajendran2, Chang Shim1, Danielle D. Jandial1, Mark R. Lackner1, Fiona Simpkins3, Funda Meric‑Bernstam4, Leslie M. Randall5, Olivier Harismendy1 1Zentalis Pharmaceuticals, Inc., San Diego, CA, USA; 2Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA; 3Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Penn Ovarian Cancer Research Center, University of Pennsylvania, Philadelphia, PA, USA; 4Department of Investigational Cancer Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX, USA; 5Massey Comprehensive Cancer Center, VCUHealth, Richmond, VA, USA. HGSOC evaluable for MR analysis (N=123) Dose at 5:2 intermittent (mg) N (%) 300 24 (19) 350 3 (2) 400 96 (78) On‑treatment timepoint N (%) C2D1 47 (38) C3D1 11 (9) C2D1 and C3D1 65 (53) Best overall response (BOR) per RECIST 1.1 N (%) Confirmed Objective Response (cOR)a 25 (20) Stable disease (SD) 71 (58) Progressive disease (PD) 26 (21) NE 1 (1) Median follow‑up, months: 8.61 Exhibit 99.2

Figure 2. Azenosertib Is a Selective WEE1 Inhibitor and Modulates WEE1‑Dependent Pathways in Cancer Cell Lines -10 -8 -6 0 0 0 0.1 0.3 1 0 0. 2 0. 5 1 2 0 0. 01 0. 02 5 0. 05 0. 1 1 2 3 4 5 6 7 8 9 10 11 Dose (µM) Dose (µM) p-CDK1 (Y15)Target engagement CDK1 Cyclin E1 Cyclin B1 pS10-HH3 pS345-CHK1 γH2AX (S139) Cleaved Caspase-3 (D175) Vinculin Lane p-TCTP (S64) TCTP WEE1 p-CDK1 (Y15) Vinculin 20 40 60 80 100 120 Log [Compound (M)] Azenosertib Azenosertib A-427 (lung) BI-2536 (PLKi) N or m al iz ed BR ET Re sp on se (% ) 0 0.1 0.3 1 Azenosertib OVCAR3 (ovarian) DDR Apoptosis Cell cycle Azenosertib BI-2536 (PLKi) A B C D A. Representation of the selective kinase IC50 of azenosertib and adavosertib determined by the FRET‑based Ź‑LYTE assay (Thermo Fisher). Data extracted from Huang et al. J Med Chem. 2021.10 B. Dose‑response binding curves for azenosertib and BI‑2536 using the PLK1 nanoBRET binding assay in HEK293 cells (Promega). C. Inhibitory activity was evaluated by quantitation of p‑CDK1 (Y15) for WEE1 and p‑TCTP (S64) for PLK1. MDA‑MD‑231 cells were synchronized in the G2/M phase with 100 nM nocodazole for 8 h prior to treatment with azenosertib or BI‑2536, in dose response for 16 h. Protein expression was detected using the JESS Simple Western™ instrument (Bio‑Techne). Lane 1 contains asynchronous cell lysate. Lanes 2‑11 cells were synchronized with nocodazole and treated with DMSO, increasing amounts of azenosertib (lanes 2‑6) or BI‑2536 (lanes 7‑11). D. A‑427 and OVCAR3 cells were treated with DMSO or increasing amounts of azenosertib (0.1, 0.3, 1 μM) for 16 h and proteins were detected from cell lysates using the ProteinSimple Jess Western blot system (Bio‑Techne). Figure 3. Azenosertib Causes DNA Damage and Premature Mitotic Entry in Cancer Cells 0.00 0.25 0.50 0.75 1.00 Fr ac ti on o f c el ls Azenosertib resistant Azenosertib sensitive KURAMOCHI MDA-MB-231 NCI-H1048 OVCAR3 0.00 0.05 0.10 0.15 0.20 0.25 DMSO Fr ac ti on o f p H H 3+ c el ls Azenosertib DMSO Azenosertib DMSO Azenosertib DMSO KURAMOCHI MDA-MB-231 NCI-H1048 OVCAR3 Azenosertib DMSO Azenosertib DMSO Azenosertib DMSO Azenosertib DMSO Azenosertib KURAMOCHI MDA-MB-231 NCI-H1048 OVCAR3 S G 2/M −2 0 2 4 −2 0 2 4 −2 0 2 4 −2 0 2 4 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 pHH3 normalized expression γH 2A x no rm al iz ed e xp re ss io n 54% 37% 52% 31% 25% 11% 46% 35% 2% 9% 1% 24% 1% 24% 2% 21% 28% 34% 34% 23% 32% 22% 29% 23% 15% 20% 13% 22% 42% 43% 23% 21% G1 Cell Cycle Phase Edu-S S G2/M G1 Cell Cycle Phase Edu-S S G2/M pHH3−γH2Ax− pHH3−γH2Ax+ pHH3+γH2Ax− pHH3+γH2Ax+ A B C D A. Cell cycle cytometric analysis of azenosertib resistant lines Kuramochi (HGSOC) and MDA‑MB‑231 (BrCa), and azenosertib sensitive lines NCI‑H1048 (NSCLC) and OVCAR3 (HGSOC) with treatment for 24 h with DMSO or 1µM azenosertib. The % distribution of the cell cycle phases (G1, S, G2/M) and replication state (Edu‑ S) across cell lines and treatment conditions are represented by colored bars. B. Prevalence of pHH3+ cells in each cell line, according to treatment and cell cycle phases. C. Distribution of azenosertib treated cells according to pHH3 (x‑axis) and γH2Ax (y‑axis) expression levels across 4 cell lines (columns) and according to their cell cycle phases (rows). Each cell pHH3 status (gray scale) and γH2Ax (blue scale) is indicated. D. Distribution of cells according to γH2Ax status in both treatment conditions (A) or in azenosertib treated cells in different cell cycle phase (B) or mitotic (C) context for each cell lines. Figure 4. Azenosertib Demonstrates Broad‑Spectrum Antitumor Activity and Tolerability Across Many Solid Tumor Models 0 5 10 15 20 25 30 -20 -10 0 10 20 Days post-treatment M ea n ∆ BW ± S EM (m m 3 ) 0 5 10 15 20 25 -20 -10 0 10 20 Days post-treatment M ea n ∆ BW ± S EM (m m 3 ) 0 10 20 30 40 -20 -10 0 10 20 Days post-treatment M ea n ∆ BW ± S EM (m m 3 ) 0 5 10 15 20 25 30 -20 -10 0 10 20 Days post-treatment M ea n ∆ BW ± S EM (m m 3 ) D os e TP 53 CC N E1 RB 1 PI K3 CA FB XW 7 KR A S BR A F ST K1 1 ZR−75−1 HCC1806 MDA−MB−231 T47D MDA−MB−436 JIMT−1 HCC1569 MDA−MB−468 HCC1937 SUM149PT HCC1428 MCF−7 HT−29 LS513 COLO 205 SW403 SW620 SW1463 LS411N LoVo SW837 SW1116 SK−MES−1 NCI−H1299 H1975 SW1573 Calu−6 NCI−H1944 NCI−H358 NCI−H23 PC−9 Calu−3 NCI−H1755 A−427 NCI−H82 DMS 53 NCI−H146 A2780 OVCAR−8 SK−OV−3 OVCAR−3 TOV−21G Miapaca−2 BxPC−3 22Rv1 LNCaP DU145 NCI−H660 Rh30 Yamato−SS SK−UT−1 AN3 CA HEC−151 HEC−59 0 50 100 150 Tumor growth inhibition (% of control) 0 5 10 15 20 25 30 0 500 1000 1500 2000 2500 0 5 10 15 20 25 0 1000 2000 3000 0 10 20 30 40 0 500 1000 1500 0 5 10 15 20 25 30 0 500 1000 1500 2000 Days post-treatment M ea n TV ± S EM (m m 3 ) M ea n TV ± S EM (m m 3 ) Days post-treatment Days post-treatment Days post-treatment 50 100 50 100 Q1 Q2 Q3 Q4 Protein expression (quartiles) Tu m or g ro w th in hi bi ti on (% o f c on tr ol ) 0 0 Tu m or g ro w th in hi bi ti on (% o f c on tr ol ) Q1 Q2 Q3 Q4 Low High Protein expression (lineage specific median) Low High Low High p27_pT198 all lineages mTOR all lineages GSK3ALPHABETA_ pS21S9 breast LKB1 lung_NSCLC SNAIL breast SK-UT-1 NCI-H660 SW1116 NCI-H146 40 50 60 70 50 100 0 50 100 0 lineage breast colorectal lung_NSCLC lung_SCLC ovary pancreas prostate soft_tissue uterus 60c 60i 80c 80i mut wt amplified deleted NA Vehicle QD Azenosertib 80 mg/kg QD A B C A. In vivo efficacy across multiple indications. The percentage of tumor growth inhibition compared with vehicle control (x‑axis) is indicated for 54 cell line xenograft models, separated across 9 histologic subtypes. For experiments with comparable dosing regimens, the median TGI across experiments was used. The treatment conditions (in mg/kg i: intermittent, c: continuous) and mutational status of selected genes are indicated next to each bar. B. Tumor growth curves for SK‑UT‑1, NCI‑H660, SW116 and NCI‑H146 models shown as mean TV ± SEM over time for mice treated with either vehicle or azenosertib. Corresponding body weight changes shown in the inset. Black dashed line is normalized to mouse weight at initiation of treatment and red dashed line represents 15% loss of body weight as a measure of drug tolerability. C. The in vitro expression of p27_T198, mTOR, LKB1/STK11, or GSK3_alpha _beta_pS21_S9 or SNAIL are associated with in vivo tumor growth inhibition in all lineages (quartile of expression) or in specific lineages (median expression). Figure 5. Azenosertib Displays Efficacy in A‑427 Tumor Model That Correlates With Pharmacodynamic Markers M ea n TV ± S EM (m m 3 ) 80 m g/ kg 40 m g/ kg V eh ic le 4 h 8 h 24 h 4 h 8 h 24 h M ea n ∆ BW ± S EM (m m 3 ) 0 5 10 15 20 25 30 0 300 600 900 Days post-treatment p-CDK1 (Y15) γH2AX ** 0 5 10 15 20 25 30 -20 -10 0 10 20 Days post-treatment 4 h 8 h 24 h -100 -50 0 50 % p- CD K1 (Y 15 ) ch an ge (r el ati ve to ve hi cl e) 4 h 8 h 24 h 0 400 800 1200 % γ H 2A X in du cti on (r el ati ve to ve hi cl e) ns ns ns # # # # ** *** ** # # # # # Vehicle QD 20 mg/kg QD 40 mg/kg QD 80 mg/kg QD Vehicle 40 mg/kg QD 80 mg/kg QD A B C A. TGI plot of NOD/SCID mice bearing A‑427 tumor cells treated orally with vehicle or azenosertib at 20 mg/kg, 40 mg/kg, or 80 mg/kg orally QD, continuously for 28 days. Data are shown as mean TV ± SEM. Statistical significance was calculated using 1‑way ANOVA. **P < .01 vs vehicle. Plot of average body weight ± SEM over time for vehicle or azenosertib treated groups. Red dotted line indicates 15% of body weight loss. B. IHC analysis of p‑CDK1 Y15 (left) and γH2AX (right) on A‑427 tumors collected at indicated time after 3 consecutive days of dosing with vehicle or azenosertib. Representative images were shown. C. Bar graph representing the quantitation of the PD markers, p‑CDK1 Y15 and γH2AX. The y‑axis shows the percentage change of the H‑score relative to vehicle (evaluated by 3 independent pathologists). Statistical significance was calculated using 2‑way ANOVA. **P < .01, ***P < .01, ns. # Comparison of treatment group and vehicle group, P < .05. Figure 6. Intermittent Dosing Schedules Improve Azenosertib Efficacy and Tolerability M ea n TV ± S EM (m m 3 ) M ea n TV ± S EM (m m 3 ) M ea n TV ± S EM (m m 3 ) γH 2A X le ve l r el ati ve to ve hi cl e co nt ro l A U C 0- 24 (μ g* h/ m L) C m ax (μg/g) M ea n TV ± S EM (m m 3 ) M ea n ∆ BW ± S EM (m m 3 ) M ea n TV ± S EM (m m 3 ) 80mg/ kg QD 40mg/ kg BID 100mg/ kg Q D 50mg/ kg BID 0 7 14 21 28 35 42 0 300 600 900 1200 Days post-treatment 0 7 14 21 28 35 42 -20 -10 0 10 20 0 5 10 15 20 25 0 500 1000 1500 2500 0 5 10 15 20 25 0 600 1200 1800 2400 5 10 15 20 25 30 0 200 400 600 1000 0 5 10 15 20 25 0 500 1000 1500 2500 0 24 48 72 96 0.0 0.5 1.0 1.5 2.0 2.5 Time posttreatment (h) 18 36 54 72 0 2 4 6 8 0 Days post-treatment Days post-treatment Days post-treatmentDays post-treatment Days post-treatment Vehicle QD 40 mg/kg QD 112 mg/kg QD, 5:2 56 mg/kg QD, 5:2 80 mg/kg QD Total Cumulated Dose 1120mg 1120mg 2240mg 2240mg Vehicle QD 60 mg/kg QD 90 mg/kg QD, 5:2 80 mg/kg QD, 5:2 Total Cumulated Dose 1260mg 1200mg 1350mg 90 mg/kg QD, 4:3 1080mg Vehicle QD 100 mg/kg QD 3:4 100 mg/kg QD 4:3 100 mg/kg QD 5:2 Vehicle QD 80 mg/kg QD, 5:2 100 mg/kg QD, 5:2 50 mg/kg BID, 5:2 40 mg/kg BID, 5:2 100 mg/kg QDx5 100 mg/kg QDx3 50 mg/kg BIDx5 Tumor AUC Plasma AUC Plasma Cmax 100 mg/kg QD, continuous 100 mg/kg QD, 5:2 Vehicle QD, continuousA B C D E A-427 OVCAR3A-427 A. TGI plot (left) of NOD/SCID mice bearing A‑427 NSCLC tumors treated with azenosertib at 100 mg/kg orally QD continuously or 5:2 intermittent schedule. Time course shows mean TV values (left) after cessation of drug treatment. Plot of mean body weight over time (right) following cessation of azenosertib treatment. B. TGI plot for NOD/SCID mice bearing A‑427 (left) and OVCAR3 (right) tumors treated with azenosertib at indicated dose and schedules. The total cumulated drug dose is indicated for each experimental arm over the 28‑day period (A‑427) and 21‑day period (OVCAR3). C. TGI plot for NOD/SCID mice bearing OVCAR3 tumors treated with azenosertib at indicated dose and schedules. Data are shown as mean TV ± SEM. Intermittent dose schedules: 5:2, 5 days on, 2 days off; 4:3, 4 days on, 3 days off; 3:4, 3 days on, 4 days off. D. Plot of γH2AX levels determined by IHC analysis from OVCAR3 tumors collected at each indicated time after 5 consecutive days of dosing with vehicle or azenosertib. The Y‑axis shows the change of the H‑score relative to vehicle (evaluated by 3 independent pathologists). E. Plasma and tumor samples were collected at the end of the efficacy study for PK analysis. Bar graph shows AUC (left Y‑axis) and Cmax (right Y‑axis) for azenosertib at drug dose treatment and schedule. Plasma Cmax is annotated by red diamond. Table 1. Predicted Human Exposure of Azenosertib From Preclinical Models OVCAR3 model (NOD/SCID mouse) Dosing (mg/kg) Frequency Cmax (ng/mL) Mouse AUC0‑24 (h*ng/mL) TGI (%)a Tumor regression (%)b Predicted human AUC (h*ng/mL)c 60 QD, continuous 3,713 21,147 71.9 NA 9,867 80 QD, 5:2 4,733 29,124 87.8 NA 13,631 90 QD, 4:3 5,673 36,870 96.1 NA 17,256 90 QD, 5:2 6,380 43,726 99.8 NA 20,465 100 QD, 3:4 5,087 41,902 94.4 NA 19,611 100 QD, 4:3 6,480 43,934 101.6 16.3 20,561 100 QD, 5:2 6,083 43,963 103.8 37.7 20,576 A‑427 model (NOD/SCID mouse) Dosing (mg/kg) Frequency Cmax (ng/mL) Mouse AUC0‑24 (h*ng/mL) TGI (%)a Tumor regression (%)b Predicted human AUC (h*ng/mL)c 20 QD, continuous 1,167 3,301 16 NA 1,557 40 QD, continuous 1,997 14,246 75 NA 6,719 80 QD, continuous 5,100 23,559 133 71 11,112 aTGI = (1 ‑ [Td – T0] / [Cd – C0]) × 100%. bTumor regression = (1 ‑ [Td / T0]) × 100%; T0 and Td, mean TV at the start or the end of treatment. cAdjusted by plasma protein binding rate of azenosertib, free fraction in human 34.4%, free fraction in mouse 16.1%. Figure 7. Azenosertib Induces Sustained Tumor Growth Inhibition and Is Well Tolerated After Treatment Cessation 0 20 40 60 80 100 120 140 0 600 1200 1800 2400 3000 3600 Days post-treatment Treatment 3360 mg 3360 mg 2400 mg 3100 mg 0 20 40 60 80 100 120 140 -20 -10 0 10 20 Days post-treatment M ea n TV ± S EM (m m 3 ) M ea n ∆ BW ± S EM (m m 3 ) Vehicle 40 mg/kg BID 80 mg/kg QD (120 mg/kg QD, 4:3) × 1 + (100 mg/kg QD, 4:3) × 5 (120 mg/kg QD, 5:2) × 1 + (100 mg/kg QD, 5:2) × 5 A B A. NOD/SCID mice were inoculated subcutaneously with A‑427 cells. When the mean TV reached ~200 mm3 (n=10/group), vehicle and azenosertib were administered for 42 days at indicated doses and schedule. Animals were monitored for tumor growth off‑treatment for the remainder of the study. Numbers close to each curve indicate the total cumulative dose during the treatment period. B. Mean body weight change of different treatment groups. All treatments were well tolerated (ΔBW≤15%). Data are shown as mean TV or body weight change ± SEM. Figure 8. Azenosertib Monotherapy Demonstrates Antitumor Activity in Patients With Solid Tumors Before Azenosertib treatment Patient information 63-yo male Metastatic colorectal cancer 61-yo male Metastatic NSCLC 50-yo female Uterine leiomyosarcoma cancer 51-yo male Renal cell carcinoma cancer 72-yo female Cervical adenocarcinoma cancer BTR (-51%) BTR (-49%) BTR (-48%) BTR (-68%) BTR (-35%) After Response Data derived from ZN‑c3‑001, first‑in‑human phase 1 clinical trial (NCT04158336). RESULTSBACKGROUND • High proliferative rates in cancer cells induce significant stress during DNA replication and mitosis, resulting in DNA damage and increased genomic instability • Replication stress can arise from the activation of oncogenes, insufficient metabolic nutrients or deoxynucleotide pools, and defects in DDR or cell cycle checkpoints. If cancer cells fail to adequately respond to DNA damage—by controlling cell cycle progression or repairing DNA lesions—genomic instability and the accumulation of abnormalities occur1‑4 • Targeted therapeutics exploit vulnerabilities in cancer cells created by DNA replicative stress and genomic instability by targeting key regulators of cell cycle checkpoints like WEE1. This approach holds promise for treating various cancer types5 • WEE1 checkpoint kinase plays a key role in DNA damage response by inhibiting CDK1 and CDK2, causing cell cycle arrest at G1/S and G2/M checkpoints to allow for repair of DNA damage and proper completion of DNA replication before mitosis6 • Inhibiting WEE1 kinase with selective inhibitors like azenosertib shows promise, particularly in cancers with compromised G1/S checkpoints, as it can lead to mitotic catastrophe and apoptosis7 • Clinical development of targeted checkpoint kinase inhibitors have faced challenges, notably hematologic toxicity. Optimizing dosing schedules is critical for developing a well‑tolerated clinically effective therapy5,8,9 Figure 1. Mechanism of Action of Azenosertib G2 M G1 S WEE1 G1/S Checkpoint G2/M Checkpoint WEE1 Cyclin CDK Cyclin CDK Azenosertib Azenosertib DNA damage increases and accumulates Normal Cell Cycle CDKs and their cyclin binding partners promote progression through the cell cycle 1 WEE1: Guardian of Genomic Integrity During DNA damage, WEE1 kinase phosphorylates and inactivates CDK/cyclins to halt the cell cycle and allow for repair 2 DNA Damage in Cancer DNA damage and loss of checkpoint regulators are key factors in the development and progression of cancer 3 Azenosertib Mechanism of Action Azenosertib inhibits WEE1 from inactivating CDK/cyclin; cells with damaged DNA continue through the cell cycle 4 Cancer Cell Death Cells that are forced to replicate with DNA damage undergo mitotic catastrophe and death 5 CONCLUSIONS • WEE1 plays a central role in regulating cell cycle checkpoints and is not restricted to a specific cancer indication. Our data indicates that inhibition of WEE1 promotes premature mitotic entry, induces DNA damage, and leads to mitotic catastrophe and cell death, offering potent antitumor activity across various cancer types • Azenosertib, a WEE1 inhibitor, demonstrates preclinical activity in a range of cancers beyond gynecologic malignancies, including colorectal, NSCLC, pancreatic, prostate, and soft tissue sarcomas • The study identifies potential predictive biomarkers for WEE1 inhibition efficacy (eg, expression of LKB1, mTOR, or p27), which may help select patients most likely to respond to treatment • Azenosertib does not significantly inhibit PLK1 in cellular assays, reducing concerns of off‑target toxicity and hematologic side effects • Our data demonstrates intermittent dosing schedules (eg, 5:2) improve drug tolerability and efficacy while minimizing toxicities • Azenosertib shows promising efficacy in heavily pretreated patients with advanced solid tumors in the ZN‑c3‑001 study (NCT04158336) a • Ongoing studies aim to refine biomarker strategy and combine WEE1 inhibition with other therapies for potentially improved clinical outcomes aStudy details outlined in Zentalis corporate presentation (January 29, 2025) and updated on the Zentalis pipeline website. References 1. Kotsantis P, et al. Cancer Discov. 2018;8(5):537‑555. 2. Visconti R, et al. J Exp Clin Cancer Res. 2016;35(1):153. 3. di Rorà AGL, et al. J Hematol Oncol. 2017;10(1):77. 4. Gaillard H, et al. Nat Rev Cancer. 2015;15(5):276‑289. 5. da Costa AABA, et al. Nat Rev Drug Discov. 2023;22(1):38‑58. 6. di Rorà AGL, et al. J Hematol Oncol. 2020;13(1):126. 7. Meng X, et al. Front Med (Lausanne). 2021;8:737951. 8. Jiang K, et al. Med Drug Discov. 2024;22:100185. 9. Ngoi NYL, et al. Nat Rev Clin Oncol. 2024;21(4):278‑293. 10. Huang PQ, et al. J Med Chem. 2021;64(17):13004‑13024. Acknowledgments This study is sponsored by Zentalis Pharmaceuticals, Inc. Editorial support for this poster was provided by Second City Science, LLC. Additional Information For more information on this study, visit www . zentalis . com Abbreviations 3:4, 3 days on, 4 days off; 4:3, 4 days on, 3 days off; 5:2; 5 days on, 2 days off; ABL1, tyrosine‑protein kinase; AUC, area under the curve; AUC0‑24, AUC over the last 24 hours; BID, twice daily; BrCa, breast cancer; BRET, bioluminescence resonance energy transfer; BTR, best tumor response; c, continuous; CDK, cyclin‑dependent kinase; Cmax, maximum serum concentration; CTG, CellTiter‑Glo; ΔBW, change in body weight; DDR, DNA damage response; DMSO, dimethyl sulfoxide; EdU, 5‑ethynyl‑2′‑deoxyuridine; FGR, FGR protein kinase; FRET, fluorescence resonance energy transfer; G1/S, GAP1/synthesis; G2/M, GAP2/mitosis; GSK3, glycogen synthase kinase 3; h, hour; γH2AX, phospho‑histone H2AX; HGSOC, high‑grade serous ovarian cancer; i, intermittent; IC50, half‑maximal inhibitory concentration; IHC, immunohistochemistry; JAK3, Janus kinase 3; JESS, Jess Automated Western Blot System; LCK, lymphocyte‑specific protein tyrosine kinase; LKB1, liver kinase 1; mTOR, mammalian target of rapamycin; mut, mutated; NA, not applicable; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; ns, not significant; NSCLC, non‑small cell lung cancer; p‑CDK, phospho‑cyclin‑dependent kinase; PD, pharmacodynamic; pHH3, phospho‑histone H3; PK, pharmacokinetics; PLKi, PLK inhibitor; p‑TCTP, phospho‑translationally controlled tumor protein; PLK1, polo‑like kinase 1; pS345‑CHK1, phospho‑checkpoint kinase 1 (Serine 345); pS10‑HH3, phospho‑histone H3 (Serine 10); QD, once daily; SCLC, small cell lung cancer; STK11, serine/threonine kinase 11; TCTP, translationally controlled tumor protein; TGI, tumor growth inhibition; TV, tumor volume; wt, wild‑type; yo, year old. PRESENTED AT: American Association for Cancer Research (AACR), April 25‑30, 2025, Chicago, Illinois, USA Poster #4208 Azenosertib Is a Potent and Selective WEE1 Kinase Inhibitor With Broad Antitumor Activity Across a Range of Solid Tumors Wen Liu1, Jianhui Ma1, Jiali Li1, Daehwan Kim1, Sangyub Kim1, Alexandra Levy1, Kimberly Blackwell1, Alejandro Recio‑Boiles2, Jennifer M. Segar2, Shirai Sen3, Deborah Doroshow4, Danielle Jandial1, Olivier Harismendy1, Stephan K. Grant1, Ahmed A. Samatar1, Mark R. Lackner1, Kimberlee M. Fischer1 1Zentalis Pharmaceuticals, Inc., San Diego, CA, USA; 2Department of Medicine, Hematology and Medical Oncology, The University of Arizona Comprehensive Cancer Center, Tucson, AZ, USA; 3NEXT Oncology, Dallas, TX, USA; 4Thoracic Oncology, Early Phase Trials Unit, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Kinase Adavosertib Azenosertib IC50 (nM) Ratioa IC50 (nM) Ratioa PLK1 3 1 227 60 LCK 47 16 447 112 JAK3 127 42 618 155 FGR 98 33 880 220 ABL1 187 62 1770 443 a Ratio= Kinase (IC50)/WEE1 (IC50) KURAMOCHI MDA‑MB‑231 NCI‑H1048 OVCAR3 Percentage of γH2Ax positive cells by treatment DMSO 1.0 1.0 1.3 2.8 Azenosertib 9.8 18.1 18.7 15 Distribution of γH2Ax positive cells across cell cycle phase (azenosertib treated), % G1 27.5 8.8 4.2 9.4 Edu– S 35.0 43.8 41.1 55.7 Edu+ S 11.2 12.6 16.8 5.6 G2/M 26.2 34.7 38.0 29.3 Distribution of G2/M cells by pHH3 and γH2Ax status (azenosertib treated), % pHH3–γH2Ax– 83.1 67.8 58.8 64.0 pHH3–γH2Ax+ 3.2 8.5 10.9 4.1 pHH3+ γH2Ax– 3.8 4.0 25.2 15.5 pHH3+ γH2Ax+ 9.9 19.7 5.1 16.3 Exhibit 99.3

Figure 2. Combination of Azenosertib with Encorafenib + Cetuximab (E+C) Demonstrates Synergy in BRAFV600E-Driven CRC Models In Vitro 0 20 40 60 80 100 % In hi bi ti on 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 0.031 0.063 0.125 0.25 0.5 0 0.063 0.125 0.25 0.5 1 0 0.031 0.063 0.125 0.25 0.5 0 0.031 0.063 0.125 0.25 0.5 0.05 0.025 0.013 0.006 0.003 0.001 8e-4 4e-4 2e-4 0 0.1 0.05 0.025 0.013 0.006 0.003 0.001 8e-4 4e-4 0 0.05 0.025 0.013 0.006 0.003 0.001 8e-4 4e-4 2e-4 0 0.05 0.025 0.013 0.006 0.003 0.001 8e-4 4e-4 2e-4 0 Azenosertib (μM) En co ra fe ni b (μ M ) Azenosertib (μM) Azenosertib (μM) Azenosertib (μM) LS411N HT-29 LS411N 2D 3D HT-29 350 nM Azenosertib 6 nM Encorafenib + 50 µg/ml cetuximab Triple combination 250 nM Azenosertib 5 nM Encorafenib + 50 µg/ml cetuximab Triple combination 350 nM Azenosertib 75 nM Encorafenib + 50 µg/ml cetuximab Triple combination 300 nM Azenosertib 2.5 nM Encorafenib + 50 µg/ml cetuximab Triple combination Synergy Score 0 -10 -20 -30 10 20 30 A B A. BRAFV600E CRC cell lines were treated with azenosertib in combination with encorafenib in a matrixed format for 3 days (2D) or 11 days (3D). Cell viability was measured by CellTiterGlo and plotted using the SynergyFinder tool.9 Loewe synergy scores are depicted, where scores ≥10 are synergistic. Doses are in µM. B. BRAFV600E CRC cell lines were treated with azenosertib in combination with encorafenib and cetuximab in triplicate for 3 days (2D) or 11 days (3D). Cell viability was measured by CellTiterGlo. Figure 3. Combination of Azenosertib With  E+C Further Suppresses Oncogenic Signaling, Dysregulates Cell Cycle Checkpoints, and Increases DNA Damage Azenosertib 6H 18H Encorafenib Cetuximab - + + - + - - + + + - - - + + - + + - + - - + + + - - - + + Azenosertib 6H 18H Encorafenib Cetuximab Vinculin γH2AX pERK1/2 pCDK1 (Y15) T-ERK1/2 T-CDK1 - + + - + - - + + + - - - + + - + + - + - - + + + - - - + + Vinculin γH2AX pERK1/2 pCDK1 (Y15) T-ERK1/2 T-CDK1 A B HT-29 LS411N A. LS411N cells were treated with DMSO, 250 nM azenosertib, 5 nM encorafenib, or 50 μg/ml cetuximab for 6 or 18 hours. B. HT‑29 cells were treated with DMSO, 200 nM azenosertib, 5 nM encorafenib, or 50 μg/ml cetuximab for 6 or 18 hours. Protein expression for the indicated markers was determined by Western blot. Figure 4. In Vivo Combination Treatment With  Azenosertib + Encorafenib (A+E) Drives Tumor Regression  Over High Dose of Either Single Agent, Indicating  Combinatorial Benefit of Dual Pathway Inhibition ** E+ C ** * * -25 0 25 50 75 100 % d el ta T /C V V: Vehicle A: Azenosertib E: Encorafenib C: Cetuximab A [80] E [60] C [15]+ E [40] E [10] E [20] E [40] E [10] E [20] E [40] E [10] E [20] E [40] A [40] + A [60] + A [80] + LS411N BALB/c nude mice bearing subcutaneous LS411N tumors were treated daily for 19 days (n=7/group). Doses are mentioned in mg/kg in brackets. % delta T/C was calculated and plotted using the formula: (Td‑T0)/(Cd‑C0) x 100, where each datapoint represents an individual animal change from baseline, relative to that of vehicle control. Significance relative to SOC E+C is indicated (*p<0.05; **p<0.005), otherwise the difference is non‑significant (2‑way repeated measures ANOVA). Figure 5. Combination of Azenosertib With E+C Results in Significant Combination Benefit and Induces  Tumor Regression in CDX Models of BRAFV600E-driven CRC In Vivo 0 7 14 21 0 500 1000 1500 Days on treatment M ea n TV ± S EM (m m 3 ) 0 500 1000 1500 0 7 14 21 Days on treatment -100 0 100 200 300 400 500 600 Δ TV a t d ay 2 0 (% ) -30% -100 0 100 200 300 500 700 -30% Δ TV a t d ay 2 0 (% ) M ea n TV ± S EM (m m 3 ) 63% TGI 69% TGI 108% TGI 88% TGI 15% TGI 101% TGI Vehicle Azenosertib 60 mg/kg QD Encorafenib 20 mg/kg QD + cetuximab 15 mg/kg BIW Azenosertib 60 mg/kg QD + encorafenib 20 mg/kg QD + cetuximab 15 mg/kg BIW Vehicle Azenosertib 60 mg/kg QD Encorafenib 15 mg/kg QD + cetuximab 15 mg/kg BIW Azenosertib 60 mg/kg QD + encorafenib 15 mg/kg QD + cetuximab 15 mg/kg BIW HT-29LS411NA B A. BALB/c nude mice bearing subcutaneous LS411N tumors were treated for 21 days (n=8/group). p<0.0001 for all comparisons to vehicle and for triplet compared to monotherapy azenosertib and E+C. B. BALB/c nude mice bearing subcutaneous HT‑29 tumors were treated for 22 days (n=8/group). p<0.0001 for E+C and triplet combinations compared to vehicle or monotherapy azenosertib. Statistics calculated using 2‑way repeated measures ANOVA. Bar graphs depict ΔTV for individual mice calculated using the formula: ([TVd – TV0] / TV0) × 100. Values below 0 indicate regression. Figure 6. Combination of Azenosertib With E+C Results in Significant Combination Benefit and  Anti‑Tumor Efficacy in BRAFV600E‑driven PDX models of CRC in vivo Days on treatment M ea n TV ± S EM (m m 3 ) Days on treatment Δ TV a t d ay 2 5 (% ) Δ TV a t d ay 5 8 (% ) M ea n TV ± S EM (m m 3 ) 49%TGI 43%TGI 80%TGI 87%TGI 51%TGI 98%TGI Days on treatment Δ TV a t d ay 2 7 (% ) M ea n TV ± S EM (m m 3 ) 65%TGI 19%TGI 102%TGI 0 5 10 15 20 25 0 500 1000 1500 2000 2500 200 0 500 800 1100 1400 -30% 0 5 10 15 20 25 30 0 500 1000 1500 2000 0 250 500 750 1000 -30% 0 15 30 45 60 0 300 600 900 1200 -100 -100 -100 -50 0 50 100 300 500 -30% Vinculin γH2AX pERK1/2 Vehicle Azenosertib Encorafenib + cetuximab Azenosertib + encorafenib + cetuximab pCDK1 (Y15) T-ERK1/2 T-CDK1 Vinculin γH2AX pERK1/2 Vehicle Azenosertib Encorafenib + cetuximab Azenosertib + encorafenib + cetuximab pCDK1 (Y15) T-ERK1/2 T-CDK1 Vehicle Azenosertib 60 mg/kg QD Encorafenib 20 mg/kg QD + cetuximab20 mg/kg BIW Azenosertib 60 mg/kg QD + encorafenib 20 mg/kg QD + cetuximab 20 mg/kg BIW Vehicle Azenosertib 60 mg/kg QD Encorafenib 20 mg/kg QD + cetuximab 15 mg/kg BIW Azenosertib 60 mg/kg QD + encorafenib 20 mg/kg QD + cetuximab 15 mg/kg BIW Vehicle Azenosertib 60 mg/kg QD Encorafenib 20 mg/kg QD + cetuximab 20 mg/kg BIW Azenosertib 60 mg/kg QD + encorafenib 20 mg/kg QD + cetuximab 20 mg/kg BIW CRC563 CRC563 CRC769 CRC769 CTG-1009 A D C B A. Athymic nude mice bearing subcutaneous CRC563 tumors were treated for 25 days (n=10/group). p<0.0001 for all comparisons to vehicle and for triplet compared to monotherapy azenosertib or E+C. B. Athymic nude mice bearing subcutaneous CRC769 tumors were treated for 28 days (n=10/group). p<0.0001 for E+C and triplet compared to vehicle, p<0.0001 for triplet compared to monotherapy azenosertib or E+C. C. Athymic nude mice bearing subcutaneous CTG‑1009 tumors were treated for 59 days (n=8/group). p<0.0001 for triplet compared to vehicle or monotherapy azenosertib, p<0.001 for monotherapy azenosertib compared to vehicle. Statistics calculated using 2‑way repeated measures ANOVA. Bar graphs depict ΔTV for individual mice calculated using the formula: ([TVd – TV0] / TV0) × 100. Values below 0 indicate regression. D. CRC563 PDX tumors (top) or CRC769 PDX tumors (bottom) were collected at 24 hours post last dose and analyzed by Western blot for the indicated proteins. Figure 7. Analysis of Phosphoproteomic Data by RPPA  Comparing E+C Against Triple Combination Shows Pathway  Changes Which May Contribute to Synergistic Activity CDK1_pT14 EphA2_pS897 FRS2−alpha_pY196 LRP6_pS1490 p90RSK_pT573 PKC−a−b−II_pT638_T641 PRAS40_pT246 cdc2_pY15 CDK1_pT14 Chk1_pS345 EphA2_pS897 ER−a_pS118 H2AX_pS139 Histone−H3_pS10 p70−S6K_pT389 S6_pS235_S236 Shc_pY317 cdc2_pY15 Chk1_pS345 EphA2_pS897 PRAS40_pT246 ACC_pS79 AMPKa_pT172 cdc2_pY15 CDK1_pT14 EphA2_pS897 Histone−H3_pS10 PRAS40_pT246 S6_pS240_S244 Shc_pY317 −2 −1 0 1 2 −2 −1 0 1 2 −2 −1 0 1 2 −2 −1 0 1 2 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 Ex pr es si on r ati o (lo g2 [t ri pl e/ D M SO ]) Ex pr es si on r ati o (lo g2 [t ri pl e/ D M SO ]) Ex pr es si on r ati o (lo g2 [t ri pl e/ D M SO ]) Ex pr es si on r ati o (lo g2 [t ri pl e/ D M SO ]) Expression ratio(log2[E+C/DMSO])Expression ratio(log2[E+C/DMSO]) Expression ratio(log2[E+C/DMSO])Expression ratio(log2[E+C/DMSO]) LS411N HT-29 6h 18 h 6h 18 h Cell lines were treated for 6 hours or 18 hours in biological duplicates with the indicated compounds. HT‑29: 350 nM azenosertib, 10 nM encorafenib, 50 µg/ml cetuximab. LS411N: 700 nM azenosertib, 8 nM encorafenib, 50 µg/ml cetuximab. Treated cells were collected and lysed, then RPPA was run at the MD Anderson Functional Proteomics RPPA Core Facility. Level 4 linear transformed data were used for analysis. Outlier samples were removed from the analysis dataset. Data are plotted as log2 fold‑change ratio of E+C (X‑axis) or triple combination (Y‑axis) to DMSO. Figure 8. RNA-Seq Reveals Hallmark Pathways Are Impacted by Addition  of Azenosertib to E+C in LS411N G2M_CHECKPOINT MYC_TARGETS_V2 MITOTIC_SPINDLE E2F_TARGETS MYC_TARGETS_V1 INTERFERON_ ALPHA_ RESPONSE KRAS_ SIGNALING_DN G2M_CHECKPOINT MYC_TARGETS_V2 MITOTIC_SPINDLE E2F_TARGETS MYC_TARGETS_V1 INTERFERON_ ALPHA_ RESPONSE KRAS_ SIGNALING_DN -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 Relative gene set enrichment score ([E+C]/DMSO) Re la ti ve g en e se t e nr ic hm en t sc or e (t ri pl e/ D M SO ) Re la ti ve g en e se t e nr ic hm en t sc or e (t ri pl e/ D M SO ) -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 Relative gene set enrichment score ([E+C]/DMSO) LS411N HT-29 Paired samples prepared for RPPA in Figure 7 were used for RNA‑seq. Single sample gene set enrichment analysis was performed. Data are plotted as log2 fold change of SOC (X‑axis) or triple combo (Y‑axis) relative to DMSO. RESULTSBACKGROUND • Approximately 10% of metastatic colorectal cancers (mCRC) harbor BRAFV600E mutations, which results in increased kinase activity and downstream MAPK signaling.1 BRAFV600E mutations are also associated with poor response to chemotherapy and poor prognosis in mCRC2 • Encorafenib, a BRAFV600E‑specific inhibitor, plus the anti‑EGFR antibody cetuximab (E+C) was approved in previously treated BRAFV600E mCRC patients.3 More recently, the Phase III BREAKWATER study (NCT04607421) brought E+C into the first‑line setting in combination with FOLFOX or FOLFIRI chemotherapy regimens in untreated BRAFV600E mCRC patients4 • E+C has demonstrated improvements in overall survival, overall response rate, and progression‑free survival in this patient population; however, the observed modest response suggests the need for further enhancement through a novel triple combination therapy • Oncogene‑driven cancers, including those with BRAF alterations, are associated with increased replication stress, double strand DNA breaks (γH2AX), etc.5‑7 Higher replication stress has not only been shown to drive acquired resistance to targeted therapies8, but also poses vulnerabilities to inhibitors of DNA damage response pathways, such as WEE1 inhibitor azenosertib • Given that BRAFV600E‑driven and acquired‑resistant tumors may have higher levels of baseline replication stress, we hypothesize that combination of azenosertib with E+C may enhance tumor growth inhibition over single agent or doublet treatment and could provide meaningful benefit for BRAFV600E mCRC patients Figure 1. Normal Cell Cycle Regulation and Azenosertib Mechanism of Action in BRAF-Mutant Cancer Cell EGFR Unchecked Oncogenic Signaling RAS V600E MEK ERK DNA damage Replication stress Proliferation Survival EGFR RAS BRAF MEK ERK Cell cycle progression Proliferation DNA damage DNA damage Phosphorylation, causing inactivation of Cyclin1/2 Normal Cell Signaling and Cell Cycle Regulation BRAF-Mutant Cancer Cell and Azenosertib G2 M G1 S G1/S Checkpoint G2/M Checkpoint Cyclin CDK2 Cyclin CDK1 Azenosertib Azenosertib WEE1 CDK2 Active/Not phosphorylated CDK1 Active/ Not phosphorylated WEE1 Cancer Cell Mitotic Catastrophe and Death DNA damage accumulates Cyclin CDK2 Cyclin CDK1 WEE1 CDK2 Inactive/ Phosphorylated CDK1 Inactive/ Phosphorylated Normal Cell Proliferation WEE1 G2 M G1 S G1/S Checkpoint G2/M Checkpoint DNA damage repaired CONCLUSIONS • In vitro combination of azenosertib with E+C resulted in synergistic anti‑cancer activity in both 2D and 3D cellular assays • The minimally efficacious combination which resulted in meaningful tumor regressions in LS411N model in vivo is 60 mg/kg of azenosertib in combination with 20 mg/kg of encorafenib, both of which are clinically relevant. Furthermore, the lack of tumor regressions observed with high doses of monotherapy azenosertib or encorafenib suggests the combinatorial activity of these 2 drugs in CRC preclinical models with BRAFV600E • Triplet combination with azenosertib and E+C resulted in significant anti‑tumor activity in multiple CDX and PDX models of BRAFV600E CRC. Further, triplet combination increases the overall response rate and depth of tumor regression when compared against azenosertib monotherapy or E+C doublet • Pharmacodynamic analysis of protein markers from both in vitro cell lines and in vivo tumor samples demonstrate expected on‑target reduction of phosphorylation signals and increased DNA damage with combination treatment • High‑throughput phosphoprotein analysis by RPPA demonstrated temporal changes in phosphorylated markers of various tumor growth and cell cycle pathways, including some which could confer resistance to the E+C doublet • RNA‑seq analysis of paired samples underscores the broad hallmark pathway changes observed by RPPA, with particular emphasis on G2/M checkpoint and mitotic pathway modulation • These data demonstrated that the combination of azenosertib with E+C enhances tumor growth inhibition over single‑agent or doublet treatment and could provide meaningful benefit for patients with BRAFV600E mCRC References 1. Davies H, et al. Nature. 2002;417:949‑954. 2. Sorbye H, et al. PLoS One. 2015;10(6):e0131046. 3. Tabernero J, et al. J Clin Oncol. 2021;39(4):273‑284. 4. Pfizer Inc. Press Release. December 20, 2024. https://www.pfizer.com/news/press‑release/ press‑release‑detail/us‑fda‑approves‑pfizers‑ braftovir‑combination‑regimen‑first. Accessed March 13, 2025. 5. Dietlein F, et al. Cell. 2015;162(1):146‑159. 6. Ali M, et al. Sci Transl Med. 2022;14(638):eabc7480. 7. Kostanstis P, et al. Cancer Discov. 2018;8(5):537‑555. 8. Salgueiro L, et al. EMBO Mol Med. 2020;12(3):e10941. 9. http://www.synergyfinderplus.org. Accessed March 13, 2025. Acknowledgments This study was sponsored by Zentalis Pharmaceuticals, Inc. Editorial support for this poster was provided by Second City Science, LLC. Additional Information For more information on this study, visit www . zentalis . com or contact mlackner @ zentalis . com. Abbreviations A, azenosertib; ACC, Acetyl‑CoA carboxylase; Akt, protein kinase B; AMPK, adenosine monophosphate‑activated protein kinase; BALB, Bagg albino; BIW, twice weekly; BRAF, v‑raf murine sarcoma viral oncogene homolog B1; C, cetuximab; cdc, cell division control; CDK, cyclin‑dependent kinase; CDX, cell line‑derived xenograft; Chk1, checkpoint kinase 1; CRC, colorectal cancer; CTG, CellTiter‑Glo; DMSO, dimethyl sulfoxide; E, encorafenib; E2F, early region 2 binding factor; EGFR, epidermal growth factor receptor; EphA2, ephrin type‑A receptor 2; ERa, estrogen receptor alpha; ERK, extracellular signal‑regulated kinase; FOLFIRI, folinic acid, 5‑fluorouracil and irinotecan; FOLFOX, folinic acid, 5‑fluorouracil and oxaliplatin; FRS2, fibroblast growth factor receptor substrate 2; γH2AX, phospho‑histone H2AX; G1/S, GAP1/synthesis; G2 / M, GAP2 / mitosis; KRAS, Kirsten rat sarcoma virus; LRP6, low density lipoprotein receptor‑related protein 6; MAPK, mitogen‑activated protein kinase; mCRC, metastatic colorectal cancer; MEK, mitogen‑activated protein kinase kinase; MYC, myelocytomatosis oncogene; pCDK1, phospho‑cyclin‑dependent kinase 1; PDX, patient‑derived xenograft; pERK1/2, phospho‑extracellular signal‑regulated kinase 1/2; PKC, protein kinase C; PRAS, proline‑rich Akt substrate; QD, once daily; RAS, rat sarcoma; RIPA, radioimmunoprecipitation assay; RPPA, reverse phase protein array; SHC, Src homology and collagen; SOC, standard of care; SEM, standard error of the mean; ΔTV, change in tumor volume; T‑CDK1, total cyclin‑dependent kinase 1; T‑ERK1/2, total extracellular signal‑regulated kinase 1/2; TV, tumor volume.PRESENTED AT: American Association for Cancer Research (AACR), April 25‑30, 2025, Chicago, Illinois, USA Poster #4730 The Selective WEE1 Inhibitor Azenosertib Shows Synergistic Anti‑Tumor Activity  With Encorafenib + Cetuximab in Multiple BRAFV600E Models Nathan M. Jameson, Harshit Shah, Blake Skrable, Mona Abed, Hooman Izadi, Nam Nguyen, Olivier Harismendy, Mark R. Lackner Zentalis Pharmaceuticals, Inc. San Diego, CA, USA Exhibit 99.4