Wee1 Inhibitor Research Articles

Abstract 2606: ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance

Abstract Inhibition of the DNA damage response (DDR) pathway is an attractive approach to target cancers cells. Unlike normal cells which relay on two cell cycle checkpoints to repair DNA damage, most tumor cells only depend on the G2 checkpoint because their G1 checkpoint is defective. Wee1 regulates the G2/M checkpoint and abrogating the G2 checkpoint by inhibiting Wee-1 in cancer cells with DNA damage can preferentially sensitize cancer cells to undergo mitotic catastrophe. We have developed and characterized ZN-c3, a potent and selective Wee-1 inhibitor with a differentiated kinase selectivity and pharmacokinetics profile. ZN-c3 was evaluated in ER-positive, triple negative, triple negative PARP inhibitor resistant, and Her-2 positive trastuzumab resistant breast cancers. In the ER-positive MCF-7 efficacy model, ZN-c3 demonstrated anti-tumor activity as a single agent as well as in combinations with a CDK4/6 inhibitor (Palbociclib) or ZN-c5, a selective estrogen receptor degrader (SERD). In the MDA-MB-436, a triple negative breast cancer tumor xenograft model, ZN-c3 demonstrated efficacy as a single agent and in combination with the PARP inhibitors Olaparib or Niraparib. We extended these combination studies by evaluating the efficacy of Niraparib in combination with ZN-c3 in patient-derived xenograft (PDX) triple negative breast tumors. ZN-c3 was efficacious as a single agent and the efficacy was enhanced in the combination with Niraparib. PARP inhibitors are effective and have been approved for treatment of some cancer types. However, most patients develop resistance to these agents. We sought to investigate whether ZN-c3 can overcome the acquired resistance to PARP inhibitors (Olaparib and Niraparib). We generated PARP inhibitor resistant breast tumor xenograft models. A significant tumor growth inhibition was observed in these resistant tumor xenograft models when treated with ZN-c3. These data suggest that ZN-c3 as a single agent can overcome the acquired PARP inhibitor resistance. Finally, we also evaluated ZN-c3 activity in a HER-2 positive trastuzumab resistant breast tumor xenograft model (JIMT-1) and ZN-c3 showed anti-tumor activity alone and in combination with trastuzumab. In summary, these results demonstrate that ZN-c3 is effective as a single agent and in combination with other targeted therapies in breast cancer models. Moreover, ZN-c3 can overcome the acquired resistance to PARP inhibitors and trastuzumab. ZN-c3 is currently in clinical trials for several indications and has demonstrated promising clinical activity and good tolerability. Citation Format: Jiali Li, Jianhui Ma, Petrus R. de Jong, Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Brant Boren, Sayee Hegde Hegde, Fernando Doñate, Ahmed A. Samatar. ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2606.

RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models.

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease.

MODL-19. Exploiting DNA damage response inhibitors to enhance radiation efficacy in medulloblastoma models

Abstract Despite treatment intensification, survival for certain high-risk subgroups of medulloblastoma (MB) such as p53 mutant SHH (SHHa) and MYC amplified Group 3 (Gr3) have remained dismal. Recently, the use of Carboplatin as radiosensitiser with craniospinal irradiation (CSI) has shown survival benefit in children with Gr3 MB. However, there is an ongoing need to discover newer, non-chemo radiosensitisers which have minimal toxicity and unequivocal benefit in high-risk MB. Radiation is a potent inducer of DNA damage in cancer cells. CHK1/2, ATR, and WEE1 kinases are critical regulators of the DNA damage response (DDR). We hypothesised that inhibition of these kinases may enhance radiation-induced cytotoxicity in MB. We utilised small-animal radiotherapy platform to deliver CSI to MB-tumour-bearing mice that closely mimic clinical settings. Here we demonstrate systematic preclinical evaluation of DDR inhibitors (DDRi) as radiosensitiser in MB models. In vitro treatment of Gr3 MB cell lines with either CHK1/2 inhibitor (CHKi, Prexasertib, AZD7762), ATR inhibitor (ATRi, AZD6738), or WEE1 inhibitor (WEE1i, AZD1775) along with radiation ceased post-radiation cell cycle arrest, prohibiting DNA repair, and increasing apoptosis. The combination also reduced the colony-forming ability of MB cells post-irradiation. In vivo, CSI and DDRi, each induced modest apoptosis in orthotopically-implanted high-risk MB mice. When these models were treated with CHKi+CSI, ATRi+CSI or WEE1i+CSI, the effect was cumulative. Importantly, addition of DDRi to CSI significantly prolonged the survival of MB-bearing mice compared to CSI alone. Here we also explored strategy of “vertical blockade” by simultaneous use of CHKi and WEE1i which when combined with CSI also prolonged survival in MB-bearing mice. Additionally, we evaluated various regimens to best deliver these novel agents with CSI, maximising effect and minimising toxicity. Our study opens up new realms of radiosensitisation with the use of DDRi in paediatric medulloblastoma and provides robust preclinical evidence for clinical translation.

Selected Articles from This Issue

Intratumoral electroporation of IL12-encoding plasmids (IT-pIL12-EP) enhances antitumor immunity in treated and distant tumors. However, IT-pIL12-EP outcomes are in part dictated by infiltrating T-cell composition, where higher proportions of cytotoxic T cells as opposed to more immunosuppressive immune cells predicts enhanced IT-pIL12-EP responsiveness. In an effort to increase proportions of activated cytotoxic T cells in IT-pIL12-EP-treated tumors, Han and colleagues supplemented IT-pIL12-EP with electroporation of plasmids encoding membrane-anchored anti-CD3 single-chain variable fragments (scFv). The authors found that including anti-CD3 scFv with IL12 enhances proliferation and interferon-γ (IFNγ) secretion in both CD8+ cytotoxic T cells and CD4+ T regulatory (Treg) cells, and that costimulation with membrane-bound anti-CD3 with IL12 reduces Treg suppression. Supplementing IT-pIL12-EP with plasmids encoding anti-CD3 scFv in vivo enhances proliferation and cytotoxicity of tumor antigen-specific T cells, leading to augmented antitumor immunity against B16-F10 melanoma and 4T1 mammary tumor cells. Treating human melanoma-infiltrating T cells with IL12 and anti-CD3 scFv enhances cytotoxic CD8+ expansion, IFNγ secretion, and PD-1 expression in vitro, suggesting IT-pIL12-EP and anti-CD3 scFv could augment PD-1 immune checkpoint inhibition responsiveness in melanoma patients.Tumor necrosis factor alpha (TNFα)-mediated cytotoxicity can be resisted via NF-κB activation and downstream survival signals in head and neck squamous cell carcinoma (HNSCC). Toxicities accompanying NF-κB signaling inhibition necessitate other methods of targeting the pathway. To search for novel targets affecting NF-κB signaling, Hu and colleagues performed a RNAi screen targeting the kinome and druggable genomic targets in a HNSCC cell line expressing NF-κB promoter response elements in tandem with a β-lactamase reporter gene. The screen revealed that G2/M checkpoint kinases WEE1 and cyclin dependent kinase 1 (CDK1) promote NF-κB signaling. WEE1 inhibition using AZD1775 disrupts WEE1 interactions with IκB kinase (IKK)α/β and RELA, reducing IKKα/β and RELA phosphorylation and NF-κB signaling. Correspondingly, AZD1775 augments TNFα-mediated HNSCC cell death, including in HNSCC radiotherapy in vivo. WEE1 and CDK1 expression levels and disease outcome correlations are dependent on human papillomavirus (HPV) infection status, suggesting HPV influences the signaling pathway. In addition to unveiling a novel signaling axis, this study presents a new potential way in which NF-κB can be therapeutically targeted in HNSCC.Decreased SIN3A expression in human breast cancer cell lines enhances tumorigenic phenotypes in vitro and tumor progression in vivo. However, how SIN3A abundance in breast tumors compares to its abundance in normal breast tissue is unknown, as is how SIN3A expression is regulated in breast tumors. To address these questions, Davenport and colleagues assessed SIN3A mRNA and corresponding protein levels in patients and validated these findings using public data sets (TCGA and METABRIC). The authors found that the SIN3A mRNA 3′ untranslated region harbors a sequence complementary to that of oncogenic miR-183, and that miR-183 silences SIN3A expression and enhances expression of genes that SIN3A transcriptionally represses. Accordingly, miR-183 expression negatively correlates with breast cancer patient survival and induces breast cancer cell migration and invasion in vitro and pulmonary metastasis in vivo through SIN3A downregulation. Overall, the novel miR-183-mediated regulation of SIN3A expression uncovered in this study could be used to inform metastatic breast cancer intervention strategies moving forward.Radiation and temozolomide (TMZ), the standard treatments for glioblastoma (GBM), are known to induce a non-proliferative, senescent phenotype in surviving GBM cells. However, GBM cells often eventually escape treatment-induced senescence, leading to residual tumor outgrowth and GBM recurrence. To assess whether recently discovered senolytic drugs can eliminate senescent GBM cells, Rahman and colleagues tested the effects of 10 senolytic drugs on 12 human GBM cell lines treated with radiation or TMZ. The authors found that senolytic drugs targeting BCL-XL, as well as siRNA-mediated BCL-XL abrogation, enhance senescent GBM cell death. The results of the study suggest that pharmacological BCL-XL inhibition could augment antitumor effects of radiation and TMZ treatment, and potentially inhibit residual GBM cell outgrowth and disease recurrence.

DIPG-40. Combined pharmacological and genetic screening to identify dependencies and combinations in ACVR1-mutant diffuse midline glioma

Somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, are found in 20-25% of DMG-H3K27 patients. Treatment of ACVR1-mutant patient-derived models with multiple chemotypes of ALK2 inhibitors (ALK2i) results in reduced cell viability in vitro and extended survival in orthotopic xenografts in vivo but, as single agents, these inhibitors were unable to achieve a complete anti-tumour response. Recently we reported that combinatorial treatment of ACVR1-mutant DIPG cells with vandetanib (RTK inhibitor) and everolimus (mTOR/ABC transporter inhibitor) was synergistic both in vitro and in vivo and was shown to be a feasible combination to trial clinically in this setting. To identify specific dependencies in ACVR1-mutant cells which may be translatable with novel synergistic drug combinations alongside ALK2i, we have implemented both candidate and unbiased drug and genetic screening approaches. Using a panel of patient-derived ACVR1-mutant and wild-type models, we identified synergy between multiple chemotypes of ALK2i (M4K2009/LDN-214117) and PI3K/mTOR (AZD8055/everolimus) and MEK inhibitors (trametinib), reflecting the common co-segregation of PIK3CA/PIK3R1 alterations in these tumours. Whole-genome CRISPR/Cas9 screening of ACVR1-mutant SU-DIPG-IV cells in combination with two ALK2i (M4K2009/LDN-193189), confirmed a specific MTOR genetic dependency, as well as for the protein phosphatase regulatory subunit PPP2R1A, known to play a role in MAPK pathway activation. Additional hits include the serine/threonine kinase PKMYT1, a negative regulator of the G2/M checkpoint via a functionally redundant phosphorylation of CDK1/CCNB1 alongside WEE1; confirmatory drug assays with the WEE1 inhibitor AZD1775 resulted in a synergistic interaction with ALK2i in ACVR1-mutant cells. Hits were integrated with DepMap using ‘gene-effect’ scores (Chronos) enabling filtering of common essential genes. Preliminary pathway enrichment analysis (MAGeCKFlute) identified ALK2i-specific vulnerabilities involving TGFB1/SMAD signalling and histone deacetylation. These data highlight functionally rational and novel combinatorial possibilities for children with ACVR1-mutant DMG, with systematic preclinical assessment required for prioritisation for the clinic.

IGNITE: A phase II signal-seeking trial of adavosertib targeting recurrent high-grade, serous ovarian cancer with cyclin E1 overexpression with and without gene amplification.

5515 Background: Cyclin E1 gene amplification and protein over-expression is a marker of platinum resistance in high grade serous ovarian, fallopian tube or primary peritoneal cancer (HGSC), and may predict response to WEE1 inhibition. Adavosertib, a WEE1 inhibitor, has demonstrated activity in unselected women with recurrent ovarian and serous endometrial cancer. We aimed to evaluate the efficacy of adavosertib in women with recurrent platinum resistant HGSC with cyclin E1 over-expression, with and without gene amplification. Methods: IGNITE is a multicentre, phase 2 trial with 2 cohorts of women with recurrent platinum resistant HGSC. Tumors were assessed for cyclin E1 protein expression by IHC and CCNE1 copy number by FISH. Patients were assigned to Cohort 1 if tumors were cyclin E1 over-expressed (H-score>50) and amplified (≥8 copies), and Cohort 2 if tumors were overexpressed and nonamplified. Patients with evaluable disease by RECIST v1.1 or GCIG CA-125 criteria were included. Adavosertib 300mg PO was given daily on days 1-5 and 8-12 of a 21-day cycle. The primary endpoint was investigator assessed clinical benefit (CB) defined as absence of progression for ≥ 18 weeks. Here we present the 18-week response data for the first 32 patients treated from Cohort 2, with a data cut-off of August 2021. Results: Between Jan-2020 and May-2021, 32 patients were accrued to Cohort 2. Median age was 62 years (range 42-77) and 84% had received ≥2 prior lines of chemotherapy. Median cyclin E1 IHC H-score was 120 and 28 patients (88%) had measurable disease by RECIST. Median number of cycles commenced was 8 (range 1-19). Overall response rate (ORR) was 53% and CB rate was 61% for all evaluable patients. Seventeen patients (53%) required a dose reduction, most commonly for neutropenia or fatigue. Seventeen patients experienced ≥Grade 3 treatment related adverse event, and 4 patients (15%) discontinued due to toxicity. Conclusions: The efficacy results in a biomarker-selected cohort of patients are promising with a higher response rate than reported in previous studies of adavosertib in unselected women with recurrent HGSC. Duration of response and progression free survival data will be presented as data matures. Clinical trial information: ACTRN12619001185156P. [Table: see text]

A phase 1 dose-escalation and -expansion study of IMP7068, a WEE1 inhibitor, in patients with advanced solid tumors.

e15052 Background: WEE1 kinase is a cell-cycle regulator that is important in DNA repair, and a validated antitumor target. IMP7068 is a potent and selective WEE1 inhibitor that has demonstrated antitumor activity in preclinical pharmacological models. A first-in-human, multicenter, open-label study was conducted to evaluate the safety, tolerability, pharmacokinetic (PK) characteristics, pharmacodynamic (PD) profiles and preliminary antitumor activity of IMP7068 in patients (pts) with advanced solid tumors. Methods: This phase 1 study included both a dose-escalation and dose-expansion stage. The dose-escalation stage was designed with 6 dose cohorts of IMP7068 at 30, 60, 120, 200, 300, and 400 mg (administered orally, once daily [QD] for 3 days, followed by 4 days off weekly for 21-day cycles). An accelerated titration design was used for the 30- and 60-mg dose levels, and an i3+3 design for all doses > 60 mg. Pts were adults (age ≥18 years) with confirmed advanced solid tumors that were refractory or intolerant to standard treatment, or for which no standard treatment exists. The objectives of the dose-escalation stage were to evaluate the safety and tolerability, PK and PD profiles, and antitumor activity (per RECIST v1.1) of IMP7068. Results: As of Jan 7, 2022, 9 pts were enrolled in the dose-escalation phase (average age, 56 years). An additional cohort of IMP7068 160 mg was recommended by the Safety Monitoring Committee based on PK data from the 120-mg cohort. Pts received escalating doses at 30 mg (n = 1), 60 mg (n = 1), 120 mg (n = 3), and 160 mg (n = 4). No dose-limiting toxicities (DLTs) were observed. Treatment-related adverse events (TRAEs) occurred in 4 pts, most frequently alanine aminotransferase (ALT) increased, aspartate aminotransferase (AST) increased, and diarrhea (all in 2 pts each). Most TRAEs were grade 1 or 2 in severity; and only 1 patient had grade 3 TRAEs (AST and ALT increased). Time to reach the maximum plasma concentration of IMP7068 was approximately 2–6 hours following administration of 30–160 mg doses, respectively. Following repeated QD administration for 3 days, the accumulation ratios for IMP7068 exposure parameters Cmax and AUC0-tau were 1.74–2.49 and 3.17–3.46, respectively, across the dose levels. The elimination half-life was approximately 10–28 hours after administration of 60–160 mg IMP7068. PD data showed that levels of phosphorylated cyclin-dependent kinase 1 decreased by ≥50% after treatment in the 160-mg cohort. Five of 8 evaluable pts had a best tumor response of stable disease (SD); 1 patient with colorectal cancer maintained a best response of SD for 22 weeks. Four pts remain on study. Conclusions: IMP7068 was well-tolerated with no DLTs, and conferred a meaningful PD effect and preliminary antitumor activity at doses of up to 160 mg in pts with advanced solid tumors. Dose escalation to establish the recommended phase 2 dose and/or maximum tolerated dose is continuing. Clinical trial information: NCT04768868.

The clinical relevance of tumor RAS/TP53 dual mutation in early and metastatic colorectal cancer (CRC).

3540 Background: The relevance of individual RAS (KRAS and NRAS) and TP53 mutations in CRC is well described, but the impact of the combination of both mutations together is less understood. RAS/TP53-mutant (mut) patients (pts) were selected in FOCUS 4-C for treatment with Adavosertib (Wee1 inhibitor) due to hypothesised sensitivity due to replication stress and loss of cell cycle checkpoint control. Initial analyses in CRC suggest that RAS/TP53-mut pts may be prognostically distinct from either mutation alone. Here we examine the impact of RAS/TP53-mut status in both early stage and metastatic CRC (mCRC). Methods: RAS and TP53 mutational status were assessed by whole gene targeted NGS or PCR in hotspot regions. Pts with RAS/TP53 status in the following studies were included after exclusion of BRAF-V600E mutants and MSI-H cases: for early-stage, QUASAR2 trial (N = 408) and an Australian community cohort (N = 654); for mCRC, FOCUS (N = 373) and FOCUS4 (N = 721). Biomarker prevalence, clinical characteristics and outcome data in the RAS/TP53-mut group were compared to pts not showing dual RAS/TP53-mut. Results: The prevalence of RAS/TP53-mut was greater in mCRC compared to early CRC (43.9% vs 25.4% respectively, p < 0.001). In early-stage (II & III) cohorts combined, RAS/TP53-mut pts were more likely to be female, have a right-sided primary tumour, and involved lymph nodes. In early CRC RAS/TP53-mut pts had worse outcome: DFS HR = 1.49[1.19-1.88], p = 0.001, and OS HR = 1.48[1.16-1.89], p = 0.001. In FOCUS, RAS/TP53-mut mCRC pts had inferior PFS with 1st line chemotherapy than not dual RAS/TP53-mut: 6.9 vs 8.6 months (HR = 1.44[1.17-1.79], p = 0.001), and also shorter post-progression survival (HR = 1.49, p = 0.001), and overall survival (14.9 vs 18.9 mths [HR = 1.60, p < 0.0005]). Consistently, during 16 weeks of induction chemotherapy for mCRC pts in FOCUS4, 27.4% of RAS/TP53-mut pts had progressive disease, compared with 18.4% in not dual RAS/TP53-mut; PFS from study registration was reduced in RAS/TP53-mut (5.3 vs 6.1 mths;HR = 1.53[1.22-1.94],p < 0.001), but no statistically significant difference in OS (13.6 vs 17.6 mths;HR = 1.27,p = 0.23). Outcomes by each of the four biomarker groups (RAS/TP53 dual mut; RASwt/TP53mut; RASmut/TP53 wt; RAS/TP53 dual wt) will be presented but in all cases the dual mut subgroup had the worst outcomes compared to the other three groups, marginally better than BRAF-V600E CRC. Conclusions: RAS/TP53 dual mutation status provides useful and readily available prognostic information in both early and mCRC, independent of MSI and BRAF status. It is associated with increased risk of recurrence in early CRC, and a higher risk of chemotherapy resistance and inferior outcomes in mCRC. Evaluation of treatment strategies in this sizeable patient group and further understanding of the underlying mechanism of poor outcomes are urgently required.

A phase 1 dose-finding and dose-expansion study evaluating the safety, tolerability, pharmacokinetics, and efficacy of a highly selective WEE1 inhibitor (Debio 0123) in adult patients with advanced solid tumors.

TPS2702 Background: Debio 0123 is an oral, highly selective inhibitor of the tyrosine kinase WEE1. WEE1 is a key regulator of cell cycle progression that modulates the activity of CDK1 (CDC2), influencing entry into mitosis. WEE1 inhibition results in G2 checkpoint abrogation, triggering mitosis with unrepaired DNA leading to cell death. In vitro and in vivo tumor models have shown Debio 0123 antitumor activity. Continuous exposure seems to be needed to maximize monotherapy efficacy in preclinical models. Preliminary data from an ongoing phase 1 study of intermittent Debio 0123 combined with carboplatin, showed a manageable safety profile, and signals of antitumor activity in patients (pts) with advanced solid tumors. We present the design of a phase 1 study (NCT05109975) of continuous Debio 0123 administered as a single agent in pts with advanced solid tumors. Methods: The study comprises a dose escalation part and a dose expansion part. The primary objective of the dose escalation part of the study is to determine the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) of continuous Debio 0123 monotherapy in adults with previously treated advanced solid tumors. Key secondary objectives include characterization of safety and tolerability, pharmacokinetics (PK), and preliminary antitumor activity (overall response rate [ORR]). Key inclusion criteria are histologically or cytologically confirmed locally advanced or metastatic solid tumors, and ECOG-PS 0 or 1. Key exclusion criteria are asymptomatic or unstable brain metastases, history of cardiac disorders, inability to swallow oral medication or abnormalities affecting drug absorption. Approximately 30 dose-limiting toxicity (DLT)-evaluable pts are anticipated to be enrolled based on an escalation with overdose control (EWOC) approach for 21-day treatment cycles. When the pts of each cohort become evaluable, a safety monitoring committee will review safety and tolerability and based on EWOC recommendations will decide the next dose level or will declare the MTD and/or RP2D. Pharmacodynamic biomarkers will be correlated with tumor response and/or PK. The dose-expansion part may start after MTD and/or RP2D determination. The primary objective of this part will be to characterize the safety and tolerability (e.g. percentage of pts with DLTs, serious adverse events or discontinuations) of Debio 0123 monotherapy at the MTD/RP2D, and to evaluate the anti-tumor activity (ORR) in pts with selected recurrent/progressive solid tumors. Key inclusion criteria are measurable disease per RECIST 1.1 and specific tumor types. Based on a Simon 2-stage design, up to 34 pts per tumor type cohort may be enrolled. Currently accrual to the dose escalation is ongoing in the United States and Switzerland. Clinical trial information: NCT05109975.

Pharmacokinetic and pharmacodynamic analysis of adavosertib in advanced ovarian cancer.

5579 Background: Adavosertib (AZD-1775) is a potent small molecule inhibitor of Wee-1, currently in clinical development. In a double-blind, placebo-controlled, phase 2 trial (NCT02151292), adavosertib and gemcitabine significantly prolonged progression-free survival (PFS) and overall survival (OS) in patients with recurrent platinum-resistant or platinum-refractory high grade serous ovarian cancer (HGSOV) compared to gemcitabine alone. We investigated whether plasma and intra-tumoral adavosertib concentrations correlated with survival in these patients. Methods: Adavosertib was administered orally on Days 1, 2, 8, 9, 15 and 16 at 175 mg per day, and gemcitabine was administered on Days 1, 8 and 15 at 1000 mg/m2 every 28 days. Serial blood samples were collected on Day 1 of cycle 1 after adavosertib administration. Tumor biopsies were taken 1-2 weeks after initiation of study treatments. Plasma and tumor adavosertib concentrations were determined using validated HPLC-MS/MS. Patients were divided into groups with plasma or tumor adavosertib concentrations above or below the biologically active concentration (BAC) of 125 ng/ml (Leijen et al, J Clin Onco 2016). Survival was described using the Kaplan-Meier method. Results: Among 61 HGSOV patients who received adavosertib, plasma samples were available in 47, and tumor samples were available in 31 patients. Among 25 non-HGSOV patients (exploratory cohort), plasma and tumor samples were available in 21 and 17 patients respectively. The mean maximum adavosertib concentration (Cmax) was 355.3 ± 120.9 ng/ml and 358.6 ± 117.9 ng/ml respectively. Cmax was above BAC in all patients. The mean tumor adavosertib concentration was 609.2 ± 1129.2 ng/ml (range: 0.47 – 5501 ng/ml) for HGSOV patients, and 964.2 ± 1611.2 ng/ml (range: 0.22 – 6116 ng/ml) for non-HGSOV patients. There was no correlation between Cmax and tumor adavosertib concentrations. In HGSOV, the median PFS was 5.8 months for patients with tumor concentrations above BAC, and 3.5 months for those with tumor concentrations below BAC (Hazard ratio (HR): 0.46, 95% confidence interval: 0.19 – 1.14, p = 0.06). No difference in PFS was seen in non-HGSOV patients according to tumor adavosertib concentration. Tumor adavosertib concentration did not correlate with OS. Conclusions: Although Cmax was above BAC in all patients, there was a high variability in tumor adavosertib concentrations. In HGSOV, higher tumor adavosertib concentration was associated with a trend towards improved PFS, but not OS. Our results indicate that the current adavosertib dosing regimen may not produce the desired concentrations in tumors for some patients, and further optimization may be needed.

A phase 1/2 dose-escalation and dose-expansion study of ZN-c3 in combination with gemcitabine in adult and pediatric subjects with relapsed or refractory osteosarcoma.

TPS11584 Background: Osteosarcoma (OS) is the most common primary bone malignancy of childhood and adolescence with 5-year survival rates of 65-70% for localized disease and < 30% for de novo metastatic disease or recurrent disease. Pooled analysis of previous phase 2 trials by the Children’s Oncology Group has determined a 4-month event-free survival (EFS) of 12%. The Wee1 kinase helps regulate DNA damage repair at the G2-M checkpoint. In the presence of DNA damage, the Wee1 kinase is activated, arresting cells in the G2 phase and preventing entry into the M phase. Inhibition of the Wee1 kinase abrogates the G2-M checkpoint, forcing cancer cells to undergo unscheduled mitosis even in the presence of DNA damage, leading to mitotic catastrophe. However, the Wee1 kinase is often upregulated in OS, preserving the G2-M checkpoint and allowing tumor growth and metastases. Additionally, up to 90% of OS tumors have alterations in p53, a critical protein in the regulation of the G1-S checkpoint, especially in relapsed or refractory cases. With a dysfunctional G1-S checkpoint, cancer cells further rely on G2-M checkpoint to repair DNA damage and preserve genomic integrity. Prior studies have demonstrated that pharmacologic inhibition of the Wee1 kinase produced cell death in OS cell lines and patient-derived xenografts. While p53 mutational status appeared to modulate efficacy of the Wee1 kinase inhibitor, activity was observed in p53 wild type, mutant and null cell lines. Combination therapy studies have also been performed, demonstrating potential synergism with gemcitabine. As expected, by precipitating DNA damage, susceptibility to inhibition of the G2-M checkpoint is further increased. Methods: NCT04833582 is an ongoing, open label, multicenter, phase 1/2 clinical trial to evaluate the activity of ZN-c3, an oral Wee1 inhibitor, in combination with gemcitabine in subjects ≥12 years and ≥40 kg, with relapsed, refractory OS. Subjects are dosed once daily, continuously with ZN-c3 and receive gemcitabine 1000 mg/m2 on days 1 and 8 of 21-day cycles. Up to 18 subjects are expected to enroll in the phase 1 portion based on a typical 3 + 3 escalation design; ̃60 subjects will be enrolled in the phase 2 portion, consisting of three stages: futility, promising clinical activity, and improved precision for clinical activity. The first two stages follow a Simon two-stage optimal design with 30 subjects, to differentiate an EFS rate at 18 weeks between 12% and 36% (which may be considered a more suitable endpoint for OS, compared with radiographic response). Tumor and skin punch biopsies are incorporated into the trial to identify potential biomarkers of treatment response. Subjects must be able to swallow oral tablets and have measurable disease by RECIST v1.1; prior exposure to gemcitabine is allowed. Global enrollment began August 1, 2021, and is ongoing. Clinical trial information: NCT04833582.

Adavosertib in combination with carboplatin in advanced TP53-mutated platinum-resistant ovarian cancer.

5516 Background: Ovarian cancer is globally the second most common cause of death among women with gynecologic malignancies. Despite high initial response rates, the overall prognosis of this patient population remains poor. The majority of advanced ovarian cancers will become platinum resistant defined by recurrence within six months after completion of platinum therapy. In the first part of the current phase II study, the combination of carboplatin and the Wee1 inhibitor adavosertib (AZD1775), showed to be safe and effective in patients with TP53 mutated platinum resistant ovarian cancer. The aim of this additional cohort is to gain more information about the safety and efficacy of the combination and to explore predictive biomarkers for resistance and response to adavosertib. Methods: In this additional cohort 29 evaluable were treated with carboplatin AUC 5 mg/ ml·min and adavosertib 225 mg BID for 2.5 days in a 21-day cycle. The anti-tumor activity was assessed according to RECIST 1.1. Pre-, on- and post-treatment biopsies were obtained to explore genetic determinants of drug resistance and response to adavosertib. Results: A total of 32 patients with a median age of 62 years (39-77 years) were enrolled in this cohort. All patients had carboplatin/paclitaxel as first line therapy. Six patients received a second-line non-platinum containing regimen. Median platinum free interval was 5.8 months (range 1.7 – 11.9). Twenty-nine patients were evaluable for efficacy. Grade 1-2 bone marrow toxicity, nausea, vomiting and fatigue were the most common adverse events. Dose reductions of carboplatin and/or adavosertib were made in 15/29 evaluable patients (52%). Dose delays occurred in the majority of patients (76%), mostly due to neutropenia and thrombocytopenia. Twelve patients showed PR as best response, resulting in an ORR of 38% in the intention-to-treat population (95% CI 21%-56%). The median PFS was 5.6 months (range 1.1-32 months, 95% CI 4.2-7.0) and median duration of response was 5.3 months (95% CI 0.13-10.5). Conclusions: Adavosertib 225 mg BID for 2.5 days and carboplatin AUC 5 in a 21-day cycle could be safely combined and shows promising anti-tumor efficacy in patients with platinum resistant ovarian cancer. Bone marrow toxicity remains the most common reason for dose reductions and dose delays. Translational biomarker results (CCNE1 analysis as potential predictive marker for response and resistance and WGS) to better understand the anti-tumor activity of the combination are pending. Clinical trial information: NCT01164995.

Inhibiting the IRE1α Axis of the Unfolded Protein Response Enhances the Antitumor Effect of AZD1775 in TP53 Mutant Ovarian Cancer.

Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA‐like ER kinase (PERK) and inositol‐required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF‐κB mediated senescence‐associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP‐homologous protein (CHOP), while IRE1α‐induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α‐XBP1s pathway may enhance the efficacy of AZD1775 in the clinics.

An isogenic cell line panel for sequence-based screening of targeted anticancer drugs

SummaryWe describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC.

SUMMARYSmall cell lung cancers (SCLCs) have high mutational burden but are relatively unresponsive to immune checkpoint blockade (ICB). Using SCLC models, we demonstrate that inhibition of WEE1, a G2/M checkpoint regulator induced by DNA damage, activates the STING-TBK1-IRF3 pathway, which increases type I interferons (IFN-α and IFN-β) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade causes remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Our study demonstrates cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC models. Combined inhibition of WEE1 plus PD-L1 blockade represents a promising immunotherapeutic approach in SCLC.

161O VIOLETTE: Randomised phase II study of olaparib (ola) + ceralasertib (cer) or adavosertib (ada) vs ola alone in patients (pts) with metastatic triple-negative breast cancer (mTNBC)

Combining DNA damage response (DDR) inhibitors to inhibit multiple DDR pathways may enhance tumour cell death. Preclinical models show synergistic antitumour effects of ola (PARP1 inhibitor) + cer (ATR inhibitor) or ada (WEE1 inhibitor). VIOLETTE (NCT03330847), a phase 2 open-label study, evaluated ola ± cer or ada as 2nd–3rd line in pts with mTNBC.

Novel Therapeutic Approaches with DNA Damage Response Inhibitors for Melanoma Treatment.

Targeted therapies against components of the mitogen-activated protein kinase (MAPK) pathway and immunotherapies, which block immune checkpoints, have shown important clinical benefits in melanoma patients. However, most patients develop resistance, with consequent disease relapse. Therefore, there is a need to identify novel therapeutic approaches for patients who are resistant or do not respond to the current targeted and immune therapies. Melanoma is characterized by homologous recombination (HR) and DNA damage response (DDR) gene mutations and by high replicative stress, which increase the endogenous DNA damage, leading to the activation of DDR. In this review, we will discuss the current experimental evidence on how DDR can be exploited therapeutically in melanoma. Specifically, we will focus on PARP, ATM, CHK1, WEE1 and ATR inhibitors, for which preclinical data as single agents, taking advantage of synthetic lethal interactions, and in combination with chemo-targeted-immunotherapy, have been growing in melanoma, encouraging the ongoing clinical trials. The overviewed data are suggestive of considering DDR inhibitors as a valid therapeutic approach, which may positively impact the future of melanoma treatment.

BMP2/SMAD pathway activation in JAK2/p53-mutant megakaryocyte/erythroid progenitors promotes leukemic transformation

AbstractLeukemic transformation (LT) of myeloproliferative neoplasm (MPN) has a dismal prognosis and is largely fatal. Mutational inactivation of TP53 is the most common somatic event in LT; however, the mechanisms by which TP53 mutations promote LT remain unresolved. Using an allelic series of mouse models of Jak2/Trp53 mutant MPN, we identify that only biallelic inactivation of Trp53 results in LT (to a pure erythroleukemia [PEL]). This PEL arises from the megakaryocyte-erythroid progenitor population. Importantly, the bone morphogenetic protein 2/SMAD pathway is aberrantly activated during LT and results in abnormal self-renewal of megakaryocyte-erythroid progenitors. Finally, we identify that Jak2/Trp53 mutant PEL is characterized by recurrent copy number alterations and DNA damage. Using a synthetic lethality strategy, by targeting active DNA repair pathways, we show that this PEL is highly sensitive to combination WEE1 and poly(ADP-ribose) polymerase inhibition. These observations yield new mechanistic insights into the process of p53 mutant LT and offer new, clinically translatable therapeutic approaches.

Combinations of ATR, Chk1 and Wee1 Inhibitors with Olaparib Are Active in Olaparib Resistant Brca1 Proficient and Deficient Murine Ovarian Cells.

Simple SummaryPoly(ADP-ribose) polymerases inhibitors (PARPis), including olaparib, have been recently approved for ovarian carcinoma treatment and PARPi resistance has already been observed in the clinics. With the aim of dissecting the molecular mechanisms of PARPi resistance, we generated olaparib resistant cells lines, both in a homologous recombination (HR)-deficient and -proficient background by continuous in vitro drug treatment. In the HR proficient background, olaparib resistance was caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in olaparib resistant HR-deficient cells, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. We found that combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR status. These new olaparib resistant models will be instrumental to screen new therapeutic options for PARPi-resistant ovarian tumors.Background. Poly(ADP-ribose) polymerases inhibitor (PARPi) have shown clinical efficacy in ovarian carcinoma, especially in those harboring defects in homologous recombination (HR) repair, including BRCA1 and BRCA2 mutated tumors. There is increasing evidence however that PARPi resistance is common and develops through multiple mechanisms. Methods. ID8 F3 (HR proficient) and ID8 Brca1-/- (HR deficient) murine ovarian cells resistant to olaparib, a PARPi, were generated through stepwise drug concentrations in vitro. Both sensitive and resistant cells lines were pharmacologically characterized and the molecular mechanisms underlying olaparib resistance. Results. In ID8, cells with a HR proficient background, olaparib resistance was mainly caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in ID8 Brca1-/- HR-deficient cells resistant to olaparib, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. Importantly, combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR cell status. Conclusion. Olaparib-resistant cell lines were generated and displayed multiple mechanisms of resistance, which will be instrumental in selecting new possible therapeutic options for PARPi-resistant ovarian tumors.

HREM, RNAseq and Cell Cycle Analyses Reveal the Role of the G2/M-Regulatory Protein, WEE1, on the Survivability of Chicken Embryos during Diapause.

Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12 °C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18 °C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them, WEE1, a negative regulator of G2/M transition, was highly expressed at 12 °C compared to 18 °C. This finding suggested that cells at 12 °C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12 °C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18 °C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD.