DNA Damage Response Inhibitors Research Articles

Genetic vulnerabilities upon inhibition of DNA damage response.

Because of essential roles of DNA damage response (DDR) in the maintenance of genomic integrity, cellular homeostasis, and tumor suppression, targeting DDR has become a promising therapeutic strategy for cancer treatment. However, the benefits of cancer therapy targeting DDR are limited mainly due to the lack of predictive biomarkers. To address this challenge, we performed CRISPR screens to search for genetic vulnerabilities that affect cells’ response to DDR inhibition. By undertaking CRISPR screens with inhibitors targeting key DDR mediators, i.e. ATR, ATM, DNAPK and CHK1, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Specifically, we identified YWHAE loss as a key determinant of sensitivity to CHK1 inhibition. We showed that KLHL15 loss protects cells from DNA damage induced by ATM inhibition. Moreover, we validated that APEX1 loss sensitizes cells to DNAPK inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results enhance the understanding of DDR pathways and will facilitate the use of DDR-targeting agents in cancer therapy.

Dual targeting of the DNA damage response pathway and BCL-2 in diffuse large B-cell lymphoma

Standard chemotherapies for diffuse large B-cell lymphoma (DLBCL), based on the induction of exogenous DNA damage and oxidative stress, are often less effective in the presence of increased MYC and BCL-2 levels, especially in the case of double hit (DH) lymphomas harboring rearrangements of the MYC and BCL-2 oncogenes, which enrich for a patient’s population characterized by refractoriness to anthracycline-based chemotherapy. Here we hypothesized that adaptive mechanisms to MYC-induced replicative and oxidative stress, consisting in DNA damage response (DDR) activation and BCL-2 overexpression, could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-positive lymphoma. We first integrated targeted gene expression profiling (T-GEP), fluorescence in situ hybridization (FISH) analysis, and characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. The presence of oxidative DNA damage biomarkers identified a poor prognosis double expresser (DE)-DLBCL subset, characterized by relatively higher BCL-2 gene expression levels and enrichment for DH lymphomas. Based on these findings, we tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo DH-DLBCL models. These data provide the rationale for precision-therapy strategies based on combined DDR and BCL-2 inhibition in DH or DE-DLBCL.

Lineage-defined leiomyosarcoma subtypes emerge years before diagnosis and determine patient survival

Leiomyosarcomas (LMS) are genetically heterogeneous tumors differentiating along smooth muscle lines. Currently, LMS treatment is not informed by molecular subtyping and is associated with highly variable survival. While disease site continues to dictate clinical management, the contribution of genetic factors to LMS subtype, origins, and timing are unknown. Here we analyze 70 genomes and 130 transcriptomes of LMS, including multiple tumor regions and paired metastases. Molecular profiling highlight the very early origins of LMS. We uncover three specific subtypes of LMS that likely develop from distinct lineages of smooth muscle cells. Of these, dedifferentiated LMS with high immune infiltration and tumors primarily of gynecological origin harbor genomic dystrophin deletions and/or loss of dystrophin expression, acquire the highest burden of genomic mutation, and are associated with worse survival. Homologous recombination defects lead to genome-wide mutational signatures, and a corresponding sensitivity to PARP trappers and other DNA damage response inhibitors, suggesting a promising therapeutic strategy for LMS. Finally, by phylogenetic reconstruction, we present evidence that clones seeding lethal metastases arise decades prior to LMS diagnosis.

Intersection of Two Checkpoints: Could Inhibiting the DNA Damage Response Checkpoint Rescue Immune Checkpoint-Refractory Cancer?

Simple SummaryImmunotherapies have revolutionized the management of advanced stage cancer; however, not all patients benefit given many cancers are resistant to immune checkpoint inhibitors. To support rapid growth, malignant cells frequently bypass the cell cycle checkpoints that normally ensure high-fidelity DNA replication prior to cell division. Therefore, cancer cells (lacking early cell cycle checkpoints) are under high replication stress and rely more heavily on the DNA damage response (DDR) for survival and growth. DDR pathways are long-recognized anti-neoplastic targets. Recently, preclinical studies have demonstrated unexpected crosstalk between these pathways and the immune system. Here, we discuss emerging evidence linking cell cycle biology and the DDR to innate and adaptive immunity. We present a rationale for why Merkel cell carcinoma could serve as a paradigm for studying DDR inhibitors as novel agents to overcome resistance to programmed cell death-1 (PD-1) pathway blockade. Metastatic cancers resistant to immunotherapy require novel management strategies. DNA damage response (DDR) proteins, including ATR (ataxia telangiectasia and Rad3-related), ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), have been promising therapeutic targets for decades. Specific, potent DDR inhibitors (DDRi) recently entered clinical trials. Surprisingly, preclinical studies have now indicated that DDRi may stimulate anti-tumor immunity to augment immunotherapy. The mechanisms governing how DDRi could promote anti-tumor immunity are not well understood; however, early evidence suggests that they can potentiate immunogenic cell death to recruit and activate antigen-presenting cells to prime an adaptive immune response. Merkel cell carcinoma (MCC) is well suited to test these concepts. It is inherently immunogenic as ~50% of patients with advanced MCC persistently benefit from immunotherapy, making MCC one of the most responsive solid tumors. As is typical of neuroendocrine cancers, dysfunction of p53 and Rb with upregulation of Myc leads to the very rapid growth of MCC. This suggests high replication stress and susceptibility to DDRi and DNA-damaging agents. Indeed, MCC tumors are particularly radiosensitive. Given its inherent immunogenicity, cell cycle checkpoint deficiencies and sensitivity to DNA damage, MCC may be ideal for testing whether targeting the intersection of the DDR checkpoint and the immune checkpoint could help patients with immunotherapy-refractory cancers.

Abstract ND07: HER2-TTC: A Targeted thorium conjugate to treat HER2 expressing cancers with potent alpha radiation

Abstract HER2-TTC (BAY 2701439) is a new representative of the Targeted Thorium Conjugates (TTCs), a novel class of radiotherapeutics for the treatment of cancer. It is comprised of the α-particle emitter thorium-227 complexed to a 3,2-HOPO chelator and conjugated to a human epidermal growth factor receptor 2 (HER2) targeted monoclonal antibody derived from trastuzumab. The targeted binding of HER2-TTC exposes tumor cells that express HER2 to a cytotoxic dose of alpha radiation. The high energy alpha-particles induce clustered double-strand DNA damage in targeted and adjacent tumor cells, which have a high likelihood to result in cell death in a cell cycle-independent fashion. Alpha particles have a short range of < 100 μm (less than 10 cell diameters), which minimizes damage to surrounding healthy tissue. The mechanism of action for TTCs is not dependent on any specific tumor type or affected by known resistance mechanisms provided that the target tumor epitope continues to be expressed. Potential combinations with inhibitors of DNA damage response (DDR) pathways may lead to synergistic anti-tumor activity. Preclinically, HER2-TTC has demonstrated substantial and selective anti-tumor activity in different models including breast, gastric, lung, bladder, colorectal cancer, with a range of HER2 expressions (IHC scores of 1+ to 3+). These included trastuzumab and T-DM1 resistant models, and models with low HER2 expression (IHC2+/ISH- or IHC1+). HER2-TTC has also demonstrated synergistic efficacy in combination with olaparib in a BRCAmut model. Preclinical data also support potential combinations with immune oncology agents, likely due to immunogenic cell death induced by alpha radiation. HER2-TTC has recently entered a First in Human study (NCT04147819) in patients with advanced tumors that express HER2. These include HER2 high expressing (IHC3+ or ISH+) breast and gastric/gastroesophageal carcinoma relapsed/refractory to trastuzumab/T-DM1, cancers with low HER2 expression, and other tumors that express HER2 such as colorectal cancer, non-small cell lung cancer, salivary, bladder and endometrial tumors. Citation Format: Jenny Karlsson. HER2-TTC: A Targeted thorium conjugate to treat HER2 expressing cancers with potent alpha radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr ND07.

Abstract 2062: Comprehensive molecular profiling of DNA damage response (DDR) deficiencies in advanced solid tumors using a real-world genomic database

Abstract Purpose: DNA damage response (DDR) deficiency is a hallmark of cancer and can be exploited with therapeutic interventions such as the use of PARP inhibitors and PD-1/PD-L1 immune checkpoint inhibitors. To understand the relationship between clinically actionable DDR genomic alterations (GA) and DDR deficiency biomarkers, we investigated the distribution of the GA in the most common DDR genes and explored the association with DDR deficiency biomarkers by utilizing the Foundation Medicine comprehensive genomic database across different cancer types. These data may provide essential information to refine and define the clinical translational strategies for innovative cancer therapies in clinical development. Methods: Molecular profiles from a total of 269,371 clinically advanced and diverse malignancies were analyzed using FoundationInsightsTM. Genomic alterations including short variants, copy number alterations and rearrangements in 35 DDR genes, more complex biomarkers (tumor mutation burden [TMB], genome-wide loss of heterozygosity [gLOH], microsatellite instability [MSI] status) and PD-L1 expression were studied. Only functional alterations (known or likely oncogenic) were analyzed. Results: DDR gene alterations are frequent and non-uniformly distributed by type and frequency across cancer types including breast, ovarian, colorectal, esophagus, biliary track, gastric, head and neck, pancreas, melanoma, lung, bladder, and prostate. TP53 is the most frequently altered DDR gene across all cancers as expected (59%), followed by ARID1A (9%), ATM (4%), SMARCA4 (3%), MDM2 (3%), BRCA2 (3%), BRCA1 (2%), CHK2 (2%), MUTYH (2%), whereas many other DDR genes are rarely altered (≤1%). Excluding TP53 from the analysis, bladder cancer has the highest cumulative rate of cases with mutations in DDR genes (46.59%), while other cancers have rates ranging between 20-40%. Further analyses including the relative distribution of TMB, MSI, gLOH and PD-L1 expression by tumor type and their association with DDR GA are ongoing. Conclusion: Identifying associations of DDR alterations and immuno-oncology associated predictive biomarkers (TMB, PD-L1) as well as assessment of signatures of genomic instability (MSI, gLOH) are critical for enabling biomarker-driven precision oncology. We systematically analyzed somatic alterations using the Foundation Medicine comprehensive genomic database and provide a comprehensive molecular profiling of DDR deficiencies in advanced solid tumors. It is anticipated that this data will collectively contribute to identify molecularly defined tumor subsets, where single and/or combination therapies such as DDR inhibitors, immunotherapies, targeted therapies etc. may result in increased clinical benefit for patients with cancer. Citation Format: Danyi Wang, Brian Elenbaas, Karthikeyan Murugesan, Lee A. Albacker, Julien Doudement, Juergen Scheuenpflug, Giuseppe Locatelli, Zheng Feng. Comprehensive molecular profiling of DNA damage response (DDR) deficiencies in advanced solid tumors using a real-world genomic database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2062.

Abstract 2043: Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies

Abstract Background:The enzyme Poly(ADP-ribose) glycohydrolase (PARG) plays an important role during the DNA damage repair process through hydrolysis of poly(ADP-ribose)(PAR) chains and accounts for approximately 90% of dePARylation activity within the cell. PAR chain synthesis is achieved by poly(ADP-ribose) polymerases (PARPs), which use donor NAD+ molecules to link repeating ADP-ribose units. The resulting PAR chains serve as a platform to recruit DNA repair proteins that are critical for single-strand break (SSB) repair. Following DNA repair PARG hydrolyzes the linkages to break down PAR chains, completing the cycle. Recent studies also found that dePARylation is necessary for the translocation of DNA repair proteins such as XRCC1 to SSB suggesting a more direct role for PARG in DNA damage repair. PARP inhibitors (PARPi), demonstrate synthetic lethality in cells with impaired homologous recombination (HR)-mediated DNA repair. Clinical studies have shown that responses to PARPi are often accompanied by high rates of resistance, generating a need for additional therapies for patients with HR-deficient tumors. The ability of PARG inhibition (PARGi) to exacerbate replication deficiencies makes it a promising therapeutic target for a broad range of cancer types with genomic instability. We therefore sought out to evaluate the effects of PARGi in HR-deficient ovarian and breast cancer models. Methods: To understand the effects of PARGi in cell growth and survival, a panel of HR-deficient cell lines and cell line derived xenografts (CDX) were exposed to PARGi alone or in combination with DNA damage response inhibitors (DDRi). In these studies, PAR chain accumulation was assessed as a marker of target engagement and PARPi was used to assess on target effects in vitro. Results: Using cellular proliferation assays and xenograft models, we find that PARGi increases the cellular levels of PAR and significantly decreases the viability of HR-deficient cancer cell lines. Furthermore, inhibition of cell proliferation by PARGi is antagonized by PARPi, which is consistent with an on-target cellular mechanism of action (MOA). In conclusion, PARGi induces significant accumulation of PAR chains and decreases cell proliferation both in vitro and in vivo in HR-deficient tumor cells. Ongoing exploratory studies will further delineate the molecular underpinning of the relationship between HR-deficiency and PARGi. Citation Format: Monah Abed, Diana Munoz, Firoz Jaipuri, Nandini Ravindran, Vidya Seshadri, Marya Liimatta, Claire Neilan, Xin Linghu, An Nguyen, Marie-Claire Wagle, Zineb Mounir. Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2043.

Abstract 1965: Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile

Abstract Genome instability initiated by DNA damage or DNA replication stress is a driver of tumor development. To ensure the accuracy of DNA replication, DNA damage response (DDR) mediated by various cell cycle checkpoints activate the DNA repair system. One such checkpoint system is the G2-M checkpoint which is involved in the repair and prevention of excessive DNA damage. A key regulator of the G2-M checkpoint is the Wee1 kinase which causes cell cycle arrest to prevent damaged cells from entering mitosis. Thus, inhibition of Wee1 should prevent G2-M checkpoint activation which would lead to unscheduled mitotic entry and induction of apoptosis. Small-molecule inhibitors of Wee1 are currently being tested in several tumor types. Preliminary results show promising clinical activity of AZD-1775, a potent inhibitor of Wee1, however, achieving an optimal dose and a schedule that is well-tolerated has been a challenge. Here, we describe the characterization of ZN-c3, a potent, selective and orally bioavailable small-molecule inhibitor of Wee1. ZN-c3 exhibits potent anti-proliferative activity across multiple tumor cell lines. Compared to AZD-1775, ZN-c3 has superior selectivity profile against a panel of kinases. In vivo, ZN-c3 induced tumor regression as a single agent in several tumor models including non-small cell lung cancer, ovarian cancer, and uterine sarcoma. In combination studies, ZN-c3 potentiates the antitumor efficacies of carboplatin and gemcitabine. The pharmacokinetic and efficacy profiles of ZN-c3 and AZD-1775 were compared in tumor xenograft models. ZN-c3 showed higher plasma and tumor exposure than AZD-1775, which correlated with more potent tumor growth inhibition than AZD-1775. In summary, ZN-c3, with its key differentiated pharmacologic and kinase selectivity profile, is a promising new generation of DDR inhibitor. ZN-c3 is currently in clinical development for the treatment of advanced solid cancers. Citation Format: Jiali Li, Brant Boren, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1965.

Abstract 1022: Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC

Abstract Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung malignancy which is primarily driven by loss of function of tumor suppressor gene TP53 and RB1 and accounts for around 13-15% of all lung cancers. For metastatic SCLC the standard-of-care, first-line therapy is combination of platinum-based therapy with etoposide or with irinotecan. Though response rate in first-line chemotherapy in SCLC is very high, relapse is almost universal. For decades topotecan, a topoisomerase-I inhibitor was the only FDA approved second-line treatment in SCLC. However, on June 15th, 2020 the FDA approved lurbinectedin for treatment of patients with metastatic SCLC with disease progression from platinum-based chemotherapy and designated it as an orphan drug. Lurbinectedin (PM01183) is a synthetic analog of the natural marine-based tetrahydroisoquinoline, trabectedin which comes from the sea-squirt species Ecteinascidia turbinate. Lurbinectedin blocks transcription by inhibiting the activity of RNA-polymerase-II and inducing its specific degradation by the ubiquitin/proteasome machinery, also inducing DNA damage. Emerging data from our group, and others supports that SCLC is transcriptionally addicted via one of three main transcription factors ASCL1 (A), NeuroD1 (N) & POU2F3 (P), which may contribute to the promising results observed in SCLC trials to date with lurbinectedin. However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin. Furthermore, little is known regarding molecular changes in SCLC cells or other tumors upon exposure to lurbinectedin. In this preclinical study we investigated the therapeutic efficacy of lurbinectedin in large number of profiled SCLC cell lines representing the four SCLC subtypes (A, N, P and I) to identify candidate markers of drug sensitivity and resistance. In 12 human-derived and 3 mouse model-derived SCLC cell lines, the majority of cell lines were highly sensitive to lurbinectedin at a very low doses (median IC50 0.52 nM, range 0.08-1.84nM). We also observed increased markers of DNA damage following treatment in sensitive cell lines (e.g., γH2AX, ChK1, RPA32) by western blot. Notably, no subtype specific difference in lurbinectedin sensitivity was observed among the four subtypes (A, N, P and I) of SCLC cells. However, cell lines with higher SLFN11 expression were more sensitive to lurbinectedin. Western blot experiments showed significant increase in phosphorylation of γH2AX, ChK1, RPA32 in lurbinectedin treated SCLC cells compared to DMSO treatment. Together our preliminary data confirm lurbinectedin as a potent treatment option for SCLC, and support a rationale for potential combinations with other DNA damaging agents. Citation Format: Kiran Kundu, Robert J. Cardnell, Li Shen, C. Allison Stewart, Kasey Cargill, Carl M. Gay, Jing Wang, Lauren A. Byers. Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1022.

IMGN779, a Next Generation CD33-Targeting ADC, Combines Effectively with Cytarabine in Acute Myeloid Leukemia (AML) Preclinical Models, Resulting in Increased DNA Damage Response, Cell Cycle Arrest and Apoptosis In Vitro and Prolonged Survival In Vivo

Introduction: IMGN779, a next generation CD33-targeting ADC, is comprised of a novel DNA alkylating IGN payload, an s-SPDB cleavable linker and a humanized anti-CD33 antibody. IMGN779 is currently in Phase I dose escalation in patients with relapsed/refractory AML (NCT02674763). With improved patient outcomes in mind, we set out to evaluate the mechanism, antileukemia efficacy and tolerability of the combination of IMGN779 and cytarabine using in vitro and in vivo human AML preclinical models.Methods: DNA damage, survival and apoptotic responses to IMGN779 and cytarabine were assessed using flow cytometry and immunoblotting techniques. In vitro combinations of the free IGN payload and either an MDM2 antagonist or CHK1/2 inhibitors were assessed for impact on cell viability at 72 hours across a panel of 12 AML cell lines. IMGN779 and cytarabine were evaluated in vivo for antitumor activity, using disseminated (Molm-13 and MV4-11) and subcutaneous (SC; EOL-1) AML xenograft models. All mice were monitored for body weight loss, clinical signs, and SC tumor volume (EOL-1). Assessment was terminated for weight loss >20%, clinical signs, or, in the EOL-1 model, when SC tumor volume reached approximately 1000 mm3.Results: Exposure to cytarabine in vitro led to a dose- and time-dependent increase in CD33 transcription and cell surface expression (up to 2-fold increase at 150nM at 72 hours) in multiple human AML cell lines, revealing a novel mechanism by which cytarabine may potentiate the CD33-dependent uptake of IMGN779 by AML cells. Importantly, the in vitro combination of IMGN779 and cytarabine led to dose-dependent, additive antileukemia activity (Figure A), and enhanced levels of cell cycle arrest, DNA damage response (p21, CHK2) and apoptosis (Casp3/PARP1 cleavage). Moreover, the mechanistic importance of p53 and CHK2 in IMGN779-mediated antileukemia activity was demonstrated further using the IGN free payload in in vitro combination assays with a panel of 12 human AML cell lines. The results of these experiments suggest strong synergy between IGN free payload and an MDM2 antagonist and between IGN free payload and CHK1/2 inhibitors, pointing to additional combination regimens for IMGN779.In mice, the combination of single dose (0.253 mg/kg) or repeat dosing of IMGN779 (0.0534 or 0.0107 mg/kg, weekly x3) and high-dose cytarabine (75 mg/kg, at near MTD, daily x5) was highly active in all three AML xenograft models, and was well tolerated, with no body weight loss or other toxicities noted. The IMGN779 doses in these highly active combinations were far below the MTD (48 mg/kg). In MV4-11 and Molm-13 (both FLT3-ITD) disseminated xenografts, the combination resulted in a highly active 24-26 day tumor growth delay and 42-110% Increased Life Span (ILS), compared to minimally active IMGN779 (15-33% ILS) and inactive cytarabine (0-0% ILS) single agents. In the EOL-1 (chemo-resistant) SC model, 0.253 mg/kg single dose IMGN779 plus cytarabine was also highly active, resulting in 4/6 long-term (>90 days) complete remissions (CRs), compared to 2/6 CRs for IMGN779 and no CRs for cytarabine single agents (Figure B). Tumor growth delay for the EOL-1 tumors that persisted was 17 days for the combination, compared to 8 and 0.5 days for IMGN779 and cytarabine, respectively, supporting a highly beneficial combination effect on survival.Conclusions: IMGN779 and cytarabine are a highly effective and well tolerated combination in preclinical models of AML. Mechanistically, the IMGN779 plus cytarabine combination increased DNA damage response, cell cycle arrest, and apoptosis in vitro, which lead to increased survival in vivo . Interestingly, cytarabine increased cell surface CD33 levels on AML cells, indicating a novel mechanism by which cytarabine may potentiate IMGN779 uptake and possibly efficacy. In addition to the favorable combination with cytarabine, our in vitro combination studies also reveal the potential of combining IMGN779 with DNA damage response inhibitors. These results support rapidly advancing the combination of IMGN779 and high dose cytarabine in clinical trials. [Display omitted] DisclosuresAdams:ImmunoGen: Employment. Kelly:ImmunoGen: Employment. McCarthy:ImmunoGen: Employment. Wilhelm:ImmunoGen: Employment. Watkins:ImmunoGen: Employment. Lanieri:ImmunoGen: Employment. Sloss:ImmunoGen: Employment. Romanelli:ImmunoGen: Employment.

Enhance the Efficacy of Doxorubicin Destabilizing the G2/M Checkpoint with a CHK1/CHK2 Inhibitor Pre-Treatment on Acute Lymphoblastic Leukemia

The anthracycline antibiotic, doxorubicin, inhibits the topoisomerase II enzyme resulting in the abrogation of DNA synthesis and in the induction of DNA damages. This compound is normally used in several chemotherapy schedules for the treatment of leukemia. From a biological point of view, it has been reported that doxorubicin-treated cells arrest in G2/M phase as a consequence of the induction of DNA damages and of the induction of the DNA damage response (DDR) pathway. The activation of the G2/M checkpoint is regulated by three kinases: the cell-cycle checkpoint kinase 1 (CHK1) and 2 (CHK2) and WEE1. The activity of these kinases, which act as important tumor-suppressors on normal cells, can protect tumor cells from genotoxic agents. During the last years several studies have showed the efficacy of selective DDR inhibitors in single agent or in combination with DNA damaging agents. On these bases, the aim of the study was to evaluate the effects of a selective CHK1/CHK2 inhibitor (hereafter CHKi) in combination with doxorubicin (hereafter Doxo) on a panel of B and T acute lymphoblastic leukemia (ALL) cell lines (NALM-6, NALM19, RPMI-8402, MOLT-4 and CCRF-CEM). To this purpose we firstly evaluate the effect of Doxo in single agent on B-/T-ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathway. Cells were treated for 24/48 hours with increasing concentration of Doxo (0.25, 0.5 and 1 uM) showing a dose-dependent reduction of the cell viability, induction of apoptosis and increment of cells arrested in S or G2/M phases of the cell cycle. No differences in term of sensitivity to Doxo were seen between ALL cell lines with wildtype or mutated TP53. Then we evaluated the chemosensitizer efficacy of the CHKi against Doxo on the panel cell lines. To this purpose cells were treated simultaneously with increasing concentration of Doxo in combination with the CHKi for 24/48 hours and then the combination index of the two compounds was evaluated using Compusy Software. Only an additive effect in term of reduction of the cell viability was seen when the two compounds were added simultaneously. Based on these results we performed different schedules of combinations firstly treating with Doxo and then with CHKi or vice versa, defining the most effective schedule when cells were treated firstly with the CHKi (1 hour) and then with Doxo (24 hours). In this experimental setting the strongest effect was seen on MOLT-4 (TP53 mutated) cell line in which the pre-treatment with the CHKi impaired the G2/M checkpoint induced, later, by Doxo. Immunoblotting analyses showed that, in MOLT-4, the effect on the cell viability was associated with an increment of DNA damage (gH2AX) which was significantly higher in the combination. Interestingly on MOLT-4 the treatment with Doxo in single agent activated the cell cycle inhibitor p21 but not in the sample treated with the combination. On NALM-6 cell line (TP53 wildtype), the treatment with Doxo alone or in combination with CHKi activated p21. This data was associated with the poor effectiveness of the combination on NALM-6 cell line (poor reduction of cell viability and of induction of gH2AX). Although further experiments should be performed, first of all to investigate the role of p21 on the G2/M checkpoint, in our opinion the combination between Doxo and CHKi could be a promising strategy for the treatment of ALL. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, HARMONY project. DisclosuresMartinelli:Johnson&Johnson: Consultancy; Pfizer: Consultancy; Ariad/Incyte: Consultancy; Roche: Consultancy; Celgene: Consultancy; Amgen: Consultancy.

DOCK2 Knockdown Mitigates Disease Progression in Murine Models Harboring a FLT3/ITD Mutation

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts in the bone marrow and other tissues. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and patients who harbor a FLT3/ITD mutation have a relatively poor prognosis.Previous work in the laboratory has demonstrated that FLT3/ITD interacts with Dedicator of Cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rac GTPases. Expression of DOCK2 is limited to hematopoietic cells, and it is also expressed in the leukemic blasts of patients with FLT3/ITD AML. Knockdown (KD) of DOCK2 results in reduced Rac1 activity, and leads to decreased survival of leukemic cells that carry a FLT3/ITD mutation. This effect is seen in both in vitro studies and a mouse xenograft model, and is enhanced by treatment with cytarabine. Initial studies have shown that the anti-leukemic effect of DOCK2 KD may, in part, be due to reduced expression of both FLT3 and key DNA Damage Response (DDR) factors. In order to further investigate the relationship between DOCK2 and FLT3/ITD in hematopoietic neoplasms, we utilized mice that harbor a spontaneous Dock2 mutation (Dock2/Hsd). This mutation results in a substantial reduction in Dock2 mRNA and protein expression in hematolymphoid cells.Flt3/ITD mice develop a myeloproliferative neoplasm (MPN) that includes greatly increased spleen size. In contrast, the spleens of progeny of Flt3/ITD mice bred with Dock2/Hsd mice were greatly reduced in size. Flow cytometric analysis of the bone marrow also demonstrated that DOCK2 KD results in at least a partial reversal of the Flt3/ITD phenotype. Flt3/ITD mice characteristically demonstrate an increased fraction of immature hematopoietic stem/progenitor cells (Lin-Sca-1+c-KIT+ cells or LSKs); however, the fraction of LSKs in Flt3/ITD-Dock2/Hsd mice is similar to that seen in the marrow of WT mice. While the Flt3/ITD-Dock2/Hsd mice continued to show a disruption in the early to late Pro-B transition similar to that described in Flt3/ITD mice, the Flt3/ITD-Dock2/Hsd mice had a small but significant increase in the late Pre-B cell population, suggesting a less stringent block in B-cell development. Additionally, as compared with Flt3/ITD mice, the bone marrow of Flt3/ITD-Dock2/Hsd mice exhibited a decreased fraction of Mac1+Gr-1+ myeloid cells with a concomitant increase in Ter119+CD41- erythroid precursors, to levels that are indistinguishable from WT mice. These findings indicate that DOCK2 KD mitigates the myeloproliferative phenotype of Flt3/ITD mice.Since experiments using FLT3/ITD cell lines have suggested that DOCK2 KD may act by reducing expression of the FLT3 protein itself and also by inhibiting the DNA Damage Response, we evaluated these factors in the Flt3/ITD-Dock2/Hsd mice. Quantitative PCR of mouse whole bone marrow revealed that the mRNA levels of the Flt3 transcription regulators Meis1 and c-Myb were decreased in the Flt3/ITD-Dock2/Hsd mice, compared with Flt3/ITD mice, and the expression of Flt3 itself was markedly decreased as well. Moreover, reduced expression of key DDR factors including Chk1, Wee1, Rad51, Pim1, MSH2, MSH6 and MLH1 was also observed in Flt3/ITD-Dock2/Hsd mice.Finally, to assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine AML model that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. The Flt3/ITD-NHD13 mice develop AML with 100% penetrance and short latency; however, the expression of Dock2/Hsd in Flt3/ITD-NHD13 mice extended the median survival of these mice from approximately 110 days to over 155 days (and counting).These findings suggest that DOCK2 may cooperate with FLT3/ITD and play an important role in disease progression of FLT3/ITD leukemia. Thus, pharmacologic inhibition of DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Additionally, since DOCK2 KD results in decreased expression of DDR factors, the addition of DDR inhibitors to conventional chemotherapy may be useful in the treatment of FLT3/ITD AML. DisclosuresNo relevant conflicts of interest to declare.

ATR Inhibition Induces CDK1-SPOP Signaling and Enhances Anti-PD-L1 Cytotoxicity in Prostate Cancer.

Despite significant benefit for other cancer subtypes, immune checkpoint blockade (ICB) therapy has not yet been shown to significantly improve outcomes for men with castration-resistant prostate cancer (CRPC). Prior data have shown that DNA damage response (DDR) deficiency, via genetic alteration and/or pharmacologic induction using DDR inhibitors (DDRi), may improve ICB response in solid tumors in part due to induction of mitotic catastrophe and innate immune activation. Discerning the underlying mechanisms of this DDRi-ICB interaction in a prostate cancer-specific manner is vital to guide novel clinical trials and provide durable clinical responses for men with CRPC. We treated prostate cancer cell lines with potent, specific inhibitors of ATR kinase, as well as with PARP inhibitor, olaparib. We performed analyses of cGAS-STING and DDR signaling in treated cells, and treated a syngeneic androgen-indifferent, prostate cancer model with combined ATR inhibition and anti-programmed death ligand 1 (anti-PD-L1), and performed single-cell RNA sequencing analysis in treated tumors. ATR inhibitor (ATRi; BAY1895433) directly repressed ATR-CHK1 signaling, activated CDK1-SPOP axis, leading to destabilization of PD-L1 protein. These effects of ATRi are distinct from those of olaparib, and resulted in a cGAS-STING-initiated, IFN-β-mediated, autocrine, apoptotic response in CRPC. The combination of ATRi with anti-PD-L1 therapy resulted in robust innate immune activation and a synergistic, T-cell-dependent therapeutic response in our syngeneic mouse model. This work provides a molecular mechanistic rationale for combining ATR-targeted agents with immune checkpoint blockade for patients with CRPC. Multiple early-phase clinical trials of this combination are underway.

Radiopotentiation Profiling of Multiple Inhibitors of the DNA Damage Response for Early Clinical Development.

Radiotherapy is an effective anticancer treatment, but combinations with targeted agents that maximize efficacy while sparing normal tissue are needed. Here, we assess the radiopotentiation profiles of DNA damage response inhibitors (DDRi) olaparib (PARP1/2), ceralasertib (ATR), adavosertib (WEE1), AZD0156 (ATM), and KU-60648 (DNA-PK). We performed a radiotherapy combination screen and assessed how drug concentration and cellular DDR deficiencies influence the radiopotentiation ability of DDRi. We pre-selected six lung cancer cell lines with different genetic/signaling aberrations (including mutations in TP53 and ATM) and assessed multiple concentrations of DDRi in combination with a fixed radiotherapy dose by clonogenic assay. The effective concentration of DDRi in radiotherapy combinations is lower than that required for single-agent efficacy. This has the potential to be exploited further in the context of DDR deficiencies to increase therapeutic index and we demonstrate that low concentrations of AZD0156 preferentially sensitized p53-deficient cells. Moreover, testing multiple concentrations of DDRi in radiotherapy combinations indicated that olaparib, ceralasertib, and adavosertib have a desirable safety profile showing moderate increases in radiotherapy dose enhancement with increasing inhibitor concentration. Small increases in concentration of AZD0156 and particularly KU-60648, however, result in steep increases in dose enhancement. Radiopotentiation profiling can inform on effective drug doses required for radiosensitization in relation to biomarkers, providing an opportunity to increase therapeutic index. Moreover, multiple concentration testing demonstrates a relationship between drug concentration and radiotherapy effect that provides valuable insights that, with future in vivo validation, can guide dose-escalation strategies in clinical trials.

Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage

BackgroundLow-risk human papillomavirus (HPV), such as types 6 and 11, is considered non-oncogenic, but these types have been detected in oral cancer tissue samples, suggesting their possible involvement in oral carcinogenesis. Because double infection of high-risk HPV and Epstein-Barr virus (EBV) is known to be involved in oral carcinogenesis, we hypothesized that low-risk HPV and EBV co-infection can transform the oral cells. To verify our hypothesis, we evaluated the transformation activity of cell lines expressing both low-risk HPV E6/E7 and EBV LMP-1.MethodsWe transduced HPV6, 11 and 16 E6/E7 genes and EBV LMP-1 gene into primary mouse embryonic fibroblasts. The cell lines were examined for indices of transformation activity such as proliferation, induction of DNA damage, resistance to apoptosis, anchorage-independent growth, and tumor formation in nude mice. To evaluate the signaling pathways involved in transformation, NF-κB and p53 activities were analyzed. We also assessed adhesion signaling molecules associated with anchorage-independent growth such as MMP-2, paxillin and Cat-1.ResultsCo-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF-κB activity and reduced p53 induction. Moreover, co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escaped from apoptosis under genotoxic condition and suppression of DNA damage response (DDR). Co-expression of low-risk HPV11 E6/E7 and EBV LMP-1 demonstrated similar results. However, it led to no malignant characteristics such as anchorage-independent growth, invasiveness and tumor formation in nude mice. Compared with the cells co-expressing high-risk HPV16 E6 and EBV LMP-1 that induce transformation, co-expression of low-risk HPV6 E6 and EBV LMP-1 was associated with low MMP-2, paxillin and Cat-1 expression.ConclusionsThe co-expression of low-risk HPV E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to increased DNA damage and suppression of DDR. Thus, double infection of low-risk HPV and EBV could lead to precancerous lesions.

G2/M Checkpoint Abrogation With Selective Inhibitors Results in Increased Chromatid Breaks and Radiosensitization of 82-6 hTERT and RPE Human Cells.

While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.

Phase II trial of olaparib in combination with ceralasertib in patients with recurrent osteosarcoma.

TPS11575 Background: Osteosarcoma is the most common primary bone tumor, occurring in children, adolescents, and young adults. In contrast to advances in treatment for most childhood cancers, there have been no significant improvements in osteosarcoma outcomes in the past 40 years. Forty percent of osteosarcoma patients will, at some point, have advanced disease which has a very poor outcome with a 5-year overall survival of approximately 20%. Genomic alterations and signatures associated with sensitivity to treatment with DNA damage response (DDR) inhibitors are observed frequently in osteosarcoma. Many osteosarcomas have a unique mutational signature (signature 3) similar to that seen in BRCA1 deficient cancer and response to PARP inhibitors has been seen in osteosarcoma cell lines. Additionally, ATRX, a protein involved in the alternative lengthening of telomeres (ALT), is often inactivated in osteosarcoma. Defects in the ALT pathway may sensitize tumor cells to ATR inhibitors and osteosarcoma cell lines have been shown to be sensitive to ATR inhibition. In vitro susceptibility of osteosarcoma cell lines to ATR and PARP inhibitors, the presence of mutations in genes involved in DDR and the presence of signature 3 in a subset of osteosarcomas serves as the basis for the development of this trial. Methods: This is an ongoing open label, multicenter, phase II clinical trial to evaluate the clinical activity of PARP inhibitor, olaparib, in combination with ATR inhibitor, ceralasertib, in 2 cohorts of patients aged 12-40 with recurrent osteosarcoma (NCT04417062). Patients with unresectable disease are enrolled into Cohort 1. Patients with resectable disease limited to the lung are enrolled into Cohort 2. Patients in both cohorts receive olaparib 300mg orally twice a day on days 1-28 and ceralasertib 160 mg orally once a day on days 1-7 of a 28-day cycle (adult maximum tolerated dose for the combination). For patients in Cohort 2, study treatment also includes surgical resection of lung metastases at protocol-specified timepoints. Patients can remain on treatment for up to 2 years if they have not progressed. For Cohort 1, the primary objective is to determine whether the combination treatment improves the 4-month event-free rate as compared to a historical benchmark from Children’s Oncology Group (COG) trials using a Simon’s two-stage design. In the first stage, ≥3 of 19 patients must be event-free at 4 months to proceed to stage 2. Enrollment into Cohort 2 continues as long as Cohort 1 enrollment is ongoing. For Cohort 2, the primary endpoint is the submission of paired pre- and post-treatment tumor samples for correlative studies. Secondary endpoints include objective response rate, event-free survival, and overall survival. Integrated correlative studies will assess tumor tissue for biomarkers of treatment response and measure circulating tumor DNA longitudinally. Enrollment began November 24, 2020 and is ongoing. Clinical trial information: NCT04417062.

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control.

Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.

Intracellular PD-L1 regulates DNA damage checkpoints and suppresses Chk1 and PARP inhibitor synthetic lethality

Abstract Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.

Interfaces between cellular responses to DNA damage and cancer immunotherapy

The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.