Chk1 Inhibitor Research Articles

Design, synthesis and evaluation of novel thienopyridazine derivatives as Chk1/2 inhibitors

In order to search for novel checkpoint kinase 1/2 (Chk1) inhibitors, we have designed and synthesized a series of new compounds incorporating thienopyridazine core. Bioevaluation showed that compounds 10j, 10i, 13e and 10o exhibited relatively good inhibitory activity. Notably, compound 10o displayed high selectivity against a panel of kinases and inhibited Chk1/2 signaling pathway stimulated by DNA damage drugs in cellular level. Molecular docking of 10o to the ATP-binding site of Chk1 kinase domain indicated the existence of polar interactions between 10o and the ATP-ribose-binding residues of Chk1. In mouse HT-29 xenografts, a synergistic effect was observed. Co-treatment by CPT-11 and 10o significantly diminished the tumor volume, indicating the great potential of 10o as a candidate of Chk1/2 inhibitor.

P53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).

Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.

Abstract PD2-01: A platform of CDK4/6 inhibitor-resistant patient-derived breast cancer organoids illuminates mechanisms of resistance and therapeutic vulnerabilities

Abstract CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have revolutionized the treatment of ER+ metastatic breast cancer (MBC), significantly prolonging survival. However, this combination is not curative, and tumors eventually acquire resistance. Following progression on this combination, patients are left with limited treatment options. A diverse array of mechanisms of resistance to CDK4/6i + antiestrogens have been described. However, laboratory models that capture this heterogeneity of resistance mechanisms are lacking. Patient-derived organoids (PDOs) provide a rapid, robust and reliable platform that recapitulates intra-tumor heterogeneity, partially mimics the cancer microenvironment, and accurately predicts drug response. We aspired to generate a platform of CDK4/6i-resistant breast cancer PDOs to serve as models for understanding acquired resistance to CDK4/6i + antiestrogens and identifying therapies to overcome resistance. We successfully established 16 PDOs out of 32 biopsies (50% efficiency) of metastates from patients with ER+ MBC progressing on CDK4/6i (palbociclib or abemaciclib) + antiestrogens (letrozole or fulvestrant; median response to combination = 9 months). Our collection includes PDOs derived from lobular (n=3) and inflammatory (n=2) breast cancers and reflects racial/ethnic diversity (50% white/not Hispanic; 18.8% Hispanic; 12.5% Black; 12.5% other/unknown). Next-gen sequencing reports were available for 10 patients from which organoids were established, revealing alterations associated with CDK4/6i and/or antiestrogen resistance, including ESR1 (n=2), HER2/ERBB2 (n=2), PTEN (n=2), CCNE1 (n=1), NF1 (n=1), and ARID1A (n=1). Furthermore, one biopsy and its derived organoid lost ER expression, and 5 harbored PIK3CA activating mutations. Thus far, we have performed targeted DNA-sequencing on 7 PDOs and found 13/15 (86.7%) concordance with driver mutations from tumor NGS reports. PDOs established from CDK4/6i-resistant biopsies maintained resistance to palbociclib or abemaciclib ± fulvestrant (500 nM each) in 3D cell viability assays (6 days of treatment). In contrast, control PDOs established from primary ER+ breast cancer surgical samples (n=2) were sensitive to each CDK4/6i ± fulvestrant (median viability for combination=25.6-31.5% for control vs 65.2-80.5% for resistant). GSEA analysis of RNA-seq data from control (n=2) and CDK4/6i-resistant (n=6) PDOs cultured in estrogen-depleted media ± 200 nM palbociclib revealed that palbociclib treatment resulted in downregulation of E2F target and G2M checkpoint signatures in control but not resistant PDOs. Next, we performed a high-throughput screen of 1,000 compounds in 3 resistant PDOs. One PDO showed exquisite sensitivity to G2/M cell cycle checkpoint components, including CDK1, PLK1, Aurora kinase, ATR, Chk1, and Wee1 inhibitors. Finally, treatment of 10 resistant PDOs with the CDK2/4/6 inhibitor PF-06873600 revealed that the CCNE1 (cyclin E1)-amplified PDO was highly sensitive (IC50=130 nM vs >1000 nM), supporting that CCNE1-amplified tumors are vulnerable to CDK2 inhibition. Conclusions: PDOs can be successfully established from ER+ MBC biopsies, maintain the resistant phenotype in culture, retain driver alterations found in tumors from which they were derived, and fail to suppress E2F targets following treatment with CDK4/6i. Therefore, these PDOs represent valuable models to understand and explore diverse mechanisms of CDK4/6i resistance and therapeutic vulnerabilities. Citation Format: Ariella B. Hanker, Sumanta Chatterjee, Yunguan Wang, Dan Ye, Dhivya R. Sudhan, Brian M. Larsen, Lauren C. Smith, Yilin Zhang, Vishal Kandagatla, Kuntal Majmudar, Ezequiel Renzulli, Saurabh Mendiratta, Kimberly Blackwell, Alana L. Welm, Sunati Sahoo, Nisha Unni, Cheryl M. Lewis, Tao Wang, Ameen A. Salahudeen, Carlos L. Arteaga. A platform of CDK4/6 inhibitor-resistant patient-derived breast cancer organoids illuminates mechanisms of resistance and therapeutic vulnerabilities [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-01.

P53 oligomerization status as an indicator of sensitivity of p53-wildtype neuroblastomas to the combination of DNA damaging agent and Chk1 inhibitor.

Neuroblastomas are one of the most common types of solid tumors in infants and children and are responsible for approximately 15% of childhood cancer deaths. Neuroblastomas rarely have mutations in p53, with less than 2% of NB containing mutations in p53, compared to up to 60% for other tumor classes. Previous studies on the therapeutic combination of a DNA damaging agent and checkpoint kinase 1 (Chk1) inhibitor have shown that DNA damage-induced cell cycle arrest can be specifically abrogated in p53-defective tumors. However, some p53-wildtype tumors have also been shown to be sensitive to this therapeutic combination, suggesting that these cells have other defects in the p53 response that can be exploited for therapeutic purposes. In the current study, we investigated the response to the combination of a DNA damaging agent (SN38) and a Chk1 inhibitor (UCN-01) of four p53-wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, SK-N-AS, and Lan-5. When the cells were treated with concentrations of SN38 ranging from 0–30 ng/ml, all four cell lines accumulated p53 which was phosphorylated on serines 15 and 20. However, only the SK-N-SH were found to activate p21waf1 and repress cyclin B. In order to assess sensitivity to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 10 ng/ml SN38 for 24 h, followed by 25 nM UCN-01 for 6 and 24 h. The SK-N-SH showed no sensitivity to UCN-01 treatment whereas the SH-SY5Y, SK-N-AS, and Lan-5 abrogated G2 arrest within 24 h. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers, so we analyzed the oligomerization status of p53 in all four cell lines using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53-wildtype MCF10A cells. The SH-SY5Y, SK-N-AS, and Lan-5 however, had extremely low to undetectable levels of dimers and tetramers. Our study also showed no cytoplasmic accumulation of p53 in these cells contrary to some previous reports. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53-wildtype tumors to the therapeutic combination of DNA damaging agent and Chk1 inhibitor.

A Review of Current treatment for Triple-Negative Breast Cancer (TNBC)

We reviewed about the Triple-negative breast cancer (TNBC) and its treatment. We collected credible data from scientific database such as google scholar and Pubmed from published literatures between 2015 to 2020. According the collected literature, TNBC is one of breast cancer type can be found in women with breast cancer. It estimated about 170.000 of TNBC cases worldwide. The ER-/PR-/HER2- TNBC is the character for TNBC, and it has the poorest prognosis among other breast cancer. The risk factor of TNBC is gender, age, breastfeeding status, and special race such as African-American and Hispanic. Besides, the BRCA-1 mutation is related to TNBC. The treatment of TNBC is including surgery and radiotherapy, chemotherapy, and targeted therapy. The surgery, including BCS (Breast-conserving surgery) or called as lumpectomy and mastectomy; chemotherapy agent sensitive to TBNC, including alkylator agents, anthracyclines, taxanes, platinum-based chemotherapy, vinca alkaloids, and antimetabolites; as well targeted therapy such as PARP1 and PARP2 inhibitor, CDK (Cyclin-dependent kinase) inhibitor, p%3 inhibitor, CHK1 inhibitor, androgen antagonist, DNMT1 (DNA Methyltransferase 1) inhibitor, Anti-EGFR, PI3K/AKT/mTOR inhibitor, and anti-VEGF. These treatments are used both in early and metastatic stage of TNBC, alone or in combination.

Repeated treatments of Capan-1 cells with PARP1 and Chk1 inhibitors promote drug resistance, migration and invasion

ABSTRACT PARP1 and Chk1 inhibitors have been shown to be synergistic in different cancer models in relatively short time treatment modes. However, the consequences of long-term/repeated treatments with the combinations in cancer models remain unclear. In this study, the synergistic cytotoxicity of their combinations in 8 tumor cell lines was confirmed in a 7-day exposure mode. Then, pancreatic Capan-1 cells were repeatedly treated with the PARP1 inhibitor olaparib, the Chk1 inhibitor rabusertib or their combination for 211–214 days, during which the changes in drug sensitivity were monitored at a 35-day interval. Unexpectedly, among the 3 treatment modes, the combination treatments resulted in the highest-grade resistance to Chk1 (~14.6 fold) and PARP1 (~420.2 fold) inhibitors, respectively. Consistently, G2/M arrest and apoptosis decreased significantly in the resulting resistant variants exposed to olaparib. All 3 resistant variants also unexpectedly obtained enhanced migratory and invasive capabilities. Moreover, the combination treatments resulted in increased migration and invasion than olaparib alone. The expression of 124 genes changed significantly in all the resistant variants. We further demonstrate that activating CXCL3-ERK1/2 signaling might contribute to the enhanced migratory capabilities rather than the acquired drug resistance. Our findings indicate that repeated treatments with the rabusertib/olaparib combination result in increased drug resistance and a more aggressive cell phenotype than those with either single agent, providing new clues for future clinical anticancer tests of PARP1 and Chk1 inhibitor combinations.

A phase IA dose-escalation study of PHI-101, a new checkpoint kinase 2 inhibitor, for platinum-resistant recurrent ovarian cancer

BackgroundPHI-101 is an orally available, selective checkpoint kinase 2 (Chk2) inhibitor. PHI-101 has shown anti-tumour activity in ovarian cancer cell lines and impaired DNA repair pathways in preclinical experiments. Furthermore, the in vivo study suggests the synergistic effect of PHI-101 through combination with PARP inhibitors for ovarian cancer treatment. The primary objective of this study is to evaluate the safety and tolerability of PHI-101 in platinum-resistant recurrent ovarian cancer.MethodsChk2 inhibitor for Recurrent EpitheliAl periToneal, fallopIan, or oVarian cancEr (CREATIVE) trial is a prospective, multi-centre, phase IA dose-escalation study. Six cohorts of dose levels are planned, and six to 36 patients are expected to be enrolled in this trial.Major inclusion criteria include ≥ 19 years with histologically confirmed epithelial ovarian cancer, fallopian tube carcinoma, or primary peritoneal cancer. Also, patients who showed disease progression during platinum-based chemotherapy or disease progression within 24 weeks from completion of platinum-based chemotherapy will be included, and prior chemotherapy lines of more than five will be excluded. The primary endpoint of this study is to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of PHI-101.DiscussionPHI-101 is the first orally available Chk2 inhibitor, expected to show effectiveness in treating recurrent ovarian cancer. Through this CREATIVE trial, DLT and MTD of this new targeted therapy can be confirmed to find the recommended dose for the phase II clinical trial. This study may contribute to developing a new combination regimen for the treatment of ovarian cancer.Trial registrationClinicalTrials.gov Identifier: NCT04678102.

Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture.

BackgroundThe BMP signaling pathway plays a key role in growth, differentiation and patterning during neural development. Recent work on the generation of a self-organization of three-dimensional retinal organoid (3D-retina) from human pluripotent stem cells (hPSCs) revealed that addition of recombinant human BMP4 (rhBMP4) promotes retinal differentiation in the early neural differentiation stage. For clinical application, efficient differentiation from hPSCs to retinal cells with minimal numbers of off-target non-retinal cells is desirable. We therefore aimed to further improve an efficient retinal differentiation method for future up-scaling of cell production.MethodshPSCs were differentiated into 3D-retina using a modified SFEBq method. The effect of rhBMP4 with or without Checkpoint kinase 1 (Chk1) inhibitor (PD407824), a modulator of BMP signaling pathway, at day 3 was compared by characterizing the differentiating 3D-retina by the use of the hPSCs and immunohistochemical analysis.ResultsThe Chk1 inhibitor treatment promoted retinal differentiation from hPSCs, in combination with low-concentration rhBMP4. Addition of a Chk1 inhibitor generated a unique type of organoid with neural retina (NR) encapsulated in retinal pigment epithelium (RPE), possibly by promoting phosphorylation of SMAD1/5/9 in the cells inside the early aggregates. We confirmed that the Chk1-inhibitor-treated hPSC-3D-retina differentiated into rod and cone photoreceptor precursors and other types of retinal neurons, in long-term culture.ConclusionsIn this study, we found that combined use of rhBMP4 and a Chk1 inhibitor cooperatively promoted retinal differentiation from hPSCs. Our new retinal differentiation method is a promising option for the stable supply and up-scaling of production of 3D-retina for future cell therapy.

Combating CHK1 resistance in triple negative breast cancer: EGFR inhibition as potential combinational therapy.

Triple negative breast cancer (TNBC) is marked by a lack of expression of the Estrogen Receptor, Progesterone Receptor, and human epidermal growth factor receptor 2. Therefore, targeted therapies are being investigated based on the expression profiles of tumors. Due to the potential for acquired and intrinsic resistance, there is a need for combination therapy to overcome resistance. In the article by Lee et al., the authors identify that, while prexasertib (a CHK1 inhibitor) lacks efficacy alone, combination with an EGFR inhibitor provides synergistic anti-tumor effects. Advances in targeted therapy for TNBC will benefit the clinical landscape for this disease, with this study initiating a new avenue of investigation.

Abstract PO-131: RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer

Abstract Breast cancer (BC) is the second most diagnosed malignant disease in women, and one of the leading causes of cancer-related deaths. Triple-negative breast cancer (TNBC) is the most aggressive and difficult to treat subtype of BC because it is highly metastatic and lacks targeted therapies. African American (AA) women have a higher death rate from BC than women of other races and ethnicities. The higher incidences of TNBCs and their aggressive growth in young AA women contributing to higher death rates indicate a biological basis for this difference. Thus, it is imperative to understand the molecular mechanisms that contribute to aggressive tumor growth in AA women, identify biomarkers to select patients who will respond to existing therapies, and develop effective therapeutics to reduce this disparity. Our studies identified that multiple TNBC cells derived from AA women are inherently chemoresistant and exhibit aggressive growth behavior compared to TNBC cells derived from European American (EA) patients. Our preliminary screenings showed that the DNA repair protein, RAD51, is overexpressed in AA TNBC patients and correlates a poor prognosis relative to EA TNBC patients. Analysis of AA and EA TNBC tumor specimens indicated the epigenetic regulation of RAD51 by promoter methylations and microRNAs. Furthermore, AA women diagnosed with TNBC, have a considerably lower incidence of germline BRCA1 mutations than women of other racial or ethnic groups. This indicates most TNBC tumors in AA patients are DNA repair proficient and have intact cell cycle checkpoint mechanisms that protect them from chemotherapy-induced DNA damage and promote therapeutic resistance. Our drug screenings identified CHK1 inhibitor, Prexasertib caused DNA repair deficiency in BRCA wild-type TNBC cells by promoting proteasome-mediated degradation of BRCA1 and RAD51 proteins. Therefore, we designed a synthetic lethality-based drug combination of Prexasertib with PARP inhibitors (PARPi) in DNA repair proficient TNBC cells. Data from our preclinical evaluations show Prexasertib and Olaparib cause increased DNA strand breaks, mitotic catastrophe, and synergistic TNBC cell lethality compared to individual drug treatments. Additionally, computational analysis of TCGA data revealed a RAD51 upregulation in TNBC tumors compared to normal breast tissues and other subtypes of BC which renders as a poor prognostic marker for these patients. Remarkably, there was an interesting discrepancy in RAD51 expression levels between different racial groupings, with AA and Asian BC patients having higher levels of RAD51 expression than Caucasian BC patients. Consistent with these observations, AA and Asian TNBC patients showed decreased survival probability. Together, our data indicate that RAD51 and its epigenetic regulators could be biomarkers for aggressive TNBC and racial disparity in BC therapeutic outcomes and suggests a novel combination therapy involving Prexasertib and Olaparib may improve prognosis and reduce racial disparity in TNBC. Citation Format: Ganesh N. Acharya, Chinnadurai Mani, Upender Manne, Komaraiah Palle. RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-131.

Interaction of Wwox with Brca1 and associated complex proteins prevents premature resection at double-strand breaks and aberrant homologous recombination

Down regulation of Wwox protein expression occurs in many cancers, contributing to insensitivity to ionizing radiation (IR) and platinum drug treatments. Patients with reduced Wwox expression in their cancer tissue show decreased overall survival following these treatments, in accord with our earlier finding that reduced Wwox protein expression in cancers is associated with changes in choice of DNA double-strand break (DSB) repair pathway. Our current investigation of mechanisms underlying the initial choice of repair by homologous recombination/single-strand annealing (HR/SSA) in Wwox-deficient cells, showed immediate DNA end-resection at DSBs following IR, abrogating initial repair by the expected non-homologous end-joining (NHEJ) pathway. Mechanisms supporting the expected choice of DSB repair by NHEJ in Wwox-sufficient cells are: 1) direct recruitment of Wwox protein binding to Brca1 through the Brca1 981PPLF984 Wwox-binding motif; 2) possible Wwox blocking of Brca1-Rad50 interaction and of Brca1 activation by Chk2 phosphorylation of Brca1 S988; 3) Wwox suppression of Brca1 interaction with the B and C complex proteins, Brip1 and CtIP, thereby delaying the process of DSB end-resection post-IR. Wwox deficiency, instead, leads to early formation of the Brca1-CtIP/MRN complex at induced DSBs, stimulating immediate post-IR end-resection. This premature resection at DNA DSBs leads to inappropriate HR/SSA repair not restricted to late S/G2 cell cycle phases, and increases mutations in genomes of radiation or platinum-resistant colonies. Prevention of premature initiation of end-resection, by combining Chk2 inhibition with IR or carboplatin treatment, successfully sensitized IR and platinum-resistant Wwox-deficient cells by synthetic lethality, but did not alter response of Wwox-sufficient cells. Our results establish Wwox as a biomarker for treatment response and provide potential targets, such as Chk2, for reversal of treatment resistance.

The cytosolic iron\u2013sulfur cluster assembly (CIA) pathway is required for replication stress tolerance of cancer cells to Chk1 and ATR inhibitors

The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron–sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.

Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1.

Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro, we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo, and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

Abstract P072: Combining ATR inhibitors with carboplatin in chemoresistant TNBC conditionally reprogrammed cells and patient-derived xenografts

Abstract Background: Breast cancer is the second leading cause of cancer related death worldwide in women and triple negative breast cancer subtype is associated with a poor prognosis. TNBC patients are treated mostly with chemotherapy. In addition to the standard of care, the DNA crosslinking agent Carboplatin is often used in the treatment of both early and metastatic TNBC. A fraction of patients presents a good response, but the majority are resistant or develop resistance to chemotherapy. Overcoming chemotherapy resistance in TNBC is a major clinical unmet need and the identification of novel treatments against chemoresistant TNBC is necessary. Method: We used conditional reprogramming to establish 5 patient-derived TNBC cell lines (CRCs) from patient-derived xenografts (PDXs) generated from chemotherapy resistant tumors. All cell lines were resistant to carboplatin, and we performed shRNA high throughput screens to identify genetic vulnerabilities that could resensitize these cells to Carboplatin. We identified ataxia telangiectasia and Rad3-related protein (ATR) as a target in one of the cell lines screened. Validation of this target was performed by targeted shRNA ATR knockdown and pharmacologically with commercially available ATR inhibitors both in vitro and in vivo. Results: We confirmed that ATR inhibition resensitizes TNBC CRC to Carboplatin with shRNA ATR. We used Ceralasertib and Elimusertib, 2 ATR inhibitors, to pharmacologically validate this hit. We tested the effect of the combination of each ATR inhibitors with carboplatin (IC25) using alamar blue assays and calculated the combination index with the Chou Talalay method in all 5 cell lines. We found the combination with Elimusertib to have a more potent synergy (Average CI=0.43) than the combination with Ceralasertib (Average CI=0.68). To further explore the mechanism of ATR inhibition in this combination we measured the key targets and observed a loss of ATR protein by western blot when the cells were exposed to the combination with Elimusertib-Carboplatin but not with Ceralasertib-Carboplatin. Pharmacological inhibition of ATR main effector Chk1 with Rabusertib or by knockdown did not recapitulate the synergy observed with Elimusertib-Carboplatin, suggesting the synergy observed is independent of Chk1 activity. Surprisingly, ATR inhibition or knockdown still modulated major cell cycle players. Finally, we performed in vivo validation using the PDX from which the cell line had been derived. Our results demonstrated that addition of low doses of Elimusertib to Carboplatin significantly delayed tumor growth compared to carboplatin treatment alone and prolonged survival in animal models. Conclusion: We highlighted the efficacy of ATR inhibition to resensitize drug resistant TNBC to Carboplatin as supported by our in vitro and in vivo results. Our data also sheds light on a potentially novel mechanism of action of the Elimusertib drug involving ATR loss of expression. Our findings imply that all ATR inhibitors are not equal, and they might be associated with different mechanisms of action. Citation Format: Juliet Guay. Combining ATR inhibitors with carboplatin in chemoresistant TNBC conditionally reprogrammed cells and patient-derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P072.

Abstract P117: Oncogenic Kit induces replication stress and induces Chk1/ATR inhibitor sensitivity in melanoma

Abstract Contrary to cutaneous melanoma (CM), Acral and mucosal melanomas (AMM’s) lack effective therapy and exact a higher mortality. Since amplifications and point mutations of KIT are common in AMM, we created a multi-stage murine cellular model of human KIT melanomas (i.e. mKitK641E lines) based on the most common KIT mutation in human melanoma (p.K642E). Compared to its vector-controlled cells (mVec), mKitK641E cells exhibit greater chromosomal aberrations, and sustain 3D spheroid forming capability and aggressive tumor growth in C57BL/6J mice. In addition, DNA replication/RNA processing and ribosomal biogenesis pathways associated with KitK641E transformation. Surprisingly, an unbiased comparative drug screen uncovered a selective vulnerability to Chk1 inhibition in the KitK641E activated cells. mKitK641E cells displayed >6-fold greater sensitivity compared to mVec cells (GI50=1.5 µM vs 10 µM) for Chk1 inhibitor PF477736 treatment. Furthermore, we proved that KitK641E induces profound DNA replication stress as evidenced by increased levels of γ-H2AX and p-RPA32Ser33 in replicating cells and defective replication fork progression. Thus, mKitK641E represents a novel model to study the biology of KIT-driven melanoma and therapy development. Citation Format: Ching-Ni Njauw, Zhenyu Ji, Hensin Tsao, Antoine Simoneau, Raj Kumar, Keith T. Flaherty, Lee Zou, Duc Minh Pham. Oncogenic Kit induces replication stress and induces Chk1/ATR inhibitor sensitivity in melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P117.

FGFR2 loss sensitizes MYCN-amplified neuroblastoma CHP134 cells to CHK1 inhibitor-induced apoptosis.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i‐sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer‐related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i‐mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor–mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN‐amplified neuroblastomas.

CHK1 inhibition exacerbates replication stress induced by IGF blockade

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

ATR/CHK1/WEE1 Dependency in SRSF2-Mutated MDS/AML

Background: Mutations in splicing factor gene SRSF2 are recurrent drivers in 5-15 % of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A key property of SRSF2 mutations is that they occur early in the pathogenesis of MDS and are therefore present in all tumor cells in a patient. This property makes targetable vulnerabilities caused by SRSF2 mutations exceptionally important as they provide a way to inhibit the whole tumor. We previously demonstrated that splicing factor mutations induce R-loop-dependent activation of ATR, rendering cells sensitive to ATR inhibition. R-loops are transcription intermediates consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. Accumulation of aberrant R-loops induces the ATR kinase, which activates the G2-M cell cycle checkpoint via CHK1- and WEE1-mediated signaling. In normal cells, activation of the G2-M checkpoint halts the cell cycle until R-loops have been resolved. When the ATR pathway is inhibited, checkpoint activation does not occur, causing cells with unresolved R-loops to proceed to mitosis, resulting in DNA damage and cell death. We, therefore, sought to assess primary human MDS/AML samples for sensitivity to perturbation of the ATR/CHK1/WEE1 pathway and identify mechanisms of resistance.Methods: Sensitivity of 147 AML patient samples to 515 oncology drugs was tested ex vivo. Bone marrow mononuclear cells were incubated with 5 concentrations of each drug for 72 h followed by measurement of cell viability by CTG assay. Somatic mutations were identified by exome sequencing of matched leukemic bone marrow and skin biopsy samples.Isogenic K562 cell line clones carrying SRSF2 P95H/L/R mutations were generated using CRISPR/Cas9 editing. The presence of SRSF2 mutation was confirmed by CRISPR-sequencing and expression by whole transcriptome RNA-sequencing. Drug sensitivity of the K562 clones with and without SRSF2 mutation was determined by incubating cells with 16 concentrations of prexasertib, SRA-737, adavosertib, or BAY-1895344, followed by determination of cell viability by the MTS assay.Results: Analysis of ex vivo drug sensitivities in AML patient samples identified vulnerability to CHK1 and WEE1 inhibition in SRSF2-mutated AML: SRSF2 mutation is associated with sensitivity to the CHK1 inhibitors prexasertib (p = 0.006) (Fig 1 A and B) and PF-00477736 (p = 0.002) and the WEE1 inhibitor adavosertib (p = 0.003).To establish whether the isogenic SRSF2-mutant K562 cell line models recapitulate known downstream aberrations associated with SRSF2 mutations in patients, we analyzed gene expression and splicing. SRSF2 contains an RNA binding domain with affinity to CCNG or GGNG exonic splicing enhancer sequences. Similar to what has been observed in patients, the K562 clones with SRSF2 mutation show reduced use of GGNG sequence motifs at skipped exons. These results demonstrate that isogenic K562 clones recapitulate known alterations caused by mutant SRSF2.To determine whether SRSF2 mutations induce sensitivity to inhibition of ATR, CHK1, and WEE1, we tested 10 isogenic SRSF2 mutant and 4 wild-type K562 clones. Cells with SRSF2 mutation show increased sensitivity to ATR/CHK1/WEE1 inhibition (Fig 1C). We found no significant difference in drug sensitivity between clones carrying SRSF2 P95H/L/R substitutions. Clones with higher SRSF2 mutant allele dosage are more sensitive (Fig 1D).We identified a subset of SRSF2 mutated AML samples that were resistant to CHK1 and WEE1 inhibition. All resistant AML have co-occurring RUNX1 mutations (Fig 1B). In AML, RUNX1 mutations are associated with therapy resistance, suggesting that these mutations contribute to drug resistance. To test whether RUNX1 mutations induce resistance to ATR/CHK1/WEE1 inhibition in SRSF2-mutant leukemia, we introduced RUNX1 loss-of-function mutations in isogenic K562 carrying SRSF2 mutations. Candidate resistance factors identified by ATAC and RNA-sequencing will be validated in functional assays.Conclusions: Our results indicate that SRSF2-mutated leukemia harbor a vulnerability to the inhibition of ATR, CHK1, and WEE1 kinases. Cell line models indicate that sensitivity is similar across mutant alleles and dependent on allelic copy number. Several ATR/CHK1 and WEE1 inhibitors are in development, and our results suggest that these compounds could be effective treatments for SRSF2-mutated MDS and AML. [Display omitted] DisclosuresGraubert: astrazeneca: Research Funding; Janssen: Research Funding; Calico: Research Funding.

A Predictive Endothelial-Leukemia Pre-Clinical Platform to Uncover Drug Vulnerabilities for Personalized Treatments

Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment.Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements.Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing.Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A).We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of “public” and “private” vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs).Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E).Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. [Display omitted] DisclosuresMelnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

Combinatorial Inhibition of Galectin-9 and CHK1 Represent a Novel Treatment Strategy for T-Cell Acute Lymphoblastic Leukemia

Due to improvements in treatment strategies, the five-year event-free survival rate for pediatric patients with acute lymphoblastic leukemia (ALL) is 90%. However, patients with relapse and refractory disease fare much worse with 5-year overall survival rates of less than 50% in patients receiving chimeric antigen receptor T-cell therapy and fewer than 20% of patients surviving after receiving hematopoietic stem cell transplantation. These dismal outcomes for patients with relapse and refractory disease highlight the need for novel treatment regimens when current therapeutic options are exhausted.T-cell acute lymphoblastic leukemia (T-ALL) accounts for around 15% and 25% of ALL cases in pediatric and adult populations, respectively. This disease is driven by various molecular changes including alterations in the epigenome due, in part, to deregulated epigenetic machinery such as the polycomb repressive complex 2 (PRC2). Despite this observation, and ongoing clinical trials determining the utility of epigenetic drugs for treating various hematological malignancies, the role of the epigenome in T-ALL pathogenesis and the efficacy of epigenetic modifying drugs as treatments for this disease is heavily understudied.Galectins are members of s-type lectins which promote diverse biological processes including adhesion, signaling, and immunosuppression. Galectin-9 (Gal-9) is an emerging therapeutic target for solid cancers and hematological malignancies given that its presence is associated with poor outcomes for multiple cancers. In unpublished studies, we have found that Gal-9 is expressed on the surface of multiple human ALL subtypes with the highest basal surface expression found on T-ALL cells. To determine how this lectin impacts the function of human T-ALL cells, we treated leukemia cells with immunoglobulin control (Ig Ctrl) or anti-Gal-9 antibody (αGal-9Ab) and assessed the impact of treatment on cell cycle progression, DNA damage, and apoptosis.We used two αGal-9Ab clones for these experiments, a commercially available antibody and LYT-200 (a proprietary antibody in Phase I clinical trials for solid tumors from PureTech Health). Treatment with the commercially available antibody, but not Ctrl Ig, increased histone 3 trimethylation (H3K2me 3/H3K4me 3) with accompanying decreases in EZH2 and RING1A protein expression in human T-ALL cell lines. Antibody-induced epigenetic changes also promoted cell cycle progression (G2M transition), DNA damage, and extensive apoptosis (>90%) in multiple human T-ALL cell lines (n>6). Importantly, LYT-200 single-agent treatment also induced cell death in human T-ALL cells, demonstrating that blocking multiple epitopes on Gal-9 is sufficient to induce T-ALL cytotoxicity. These results highlight a previously unreported role for Galectin-9 in the epigenetic regulation and survival of human T-ALL cells.Given our observations that epigenome stability is critical for the survival of human T-ALL cells, we next sought to determine if the combination of αGal-9Ab treatment and epigenetic modifying drugs would further enhance the cytotoxicity of human T-ALL cells. We tested the combination of αGal-9Ab treatment and multiple drugs targeting either histone acetylation, methylation, or phosphorylation. Of these, we found that combining αGal-9Ab and GDC-0575 (a CHK1 inhibitor) resulted in extensive DNA damage and cytotoxicity (>98%). Mechanistically, we found αGal-9Ab treatment induces DNA damage in multiple human T-ALL lines, which leads to CHK1 activation. Given that GDC-0575 inhibits CHK1 activity, and CHK1 is a master regulator of the DNA damage response, we predict that the enhanced cytotoxicity of human T-ALL cells treated with the combination therapy results from the inability to effectively repair DNA damage induced by αGal-9Ab treatment. Our findings describe a previously unrecognized role for Gal-9 in T-ALL pathogenesis and demonstrates the cytotoxic effects αGal-9Ab treatment (including LYT-200) in preclinical models of human T-ALL. DisclosuresLee: PureTech Health: Research Funding. Filipovic: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.