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Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. The clinical success of PARP inhibitors has highlighted the potential of targeting these dependencies therapeutically to induce synthetic lethality. We have developed novel RAD51 inhibitors to selectively target cancers that have elevated levels of DNA damage and/or reduced levels of DNA repair. RAD51 is a critical component of the homologous recombination (HR) pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown anti-cancer activity of RAD51 inhibitors in preclinical xenograft mouse models of lymphomas that have elevated rates of DNA mutations due to aberrant expression of Activation Induced Cytidine Deaminase (AID). Breast cancer, due to high rates of genomic instability, may potentially be treated in this fashion. Here we present the characterization of our RAD51 inhibitors in breast cancer. In a murine cell line model of metastatic breast cancer, 4T1, treatment with CYT-0851 resulted in a reduction in homologous recombination activity in a sister chromatid exchange (SCE) assay at a similar rate observed in a human derived cell line (HEK293T) suggesting conservation of mechanism in human and murine models. In the same cell line, treatment with CYT-0851 and a DNA damaging agent, carboplatin, resulted in cellular toxicity above what was observed with CYT-0851, indicating that RAD51 inhibition coupled with genomic instability is cytotoxic in this breast cancer model. PARP inhibitors have been shown to induce genomic instability as measured by an increase in HR activity in SCE assays. We therefore hypothesized that our RAD51 inhibitor could act as a sensitizer to PARP inhibitors. Combinatorial treatment of 4T1 cells with CYT-0851 and olaparib reduced homologous recombination activity in the SCE assay. Additionally, combination therapy led to increased accumulation of gH2AX foci after ionizing radiation, and increased tail moment on neutral Comet assay. Chou-Talalay synergy assay demonstrated synergistic activity between olaparib and CYT-0851 in 4T1. To determine potential drug synergy in human models, a matrix study was performed with CYT-0851 and olaparib in three cell lines of triple negative breast cancer (HCC1937, HCC1143, and BT20). Concentration-dependent synergy was observed in all cell lines tested. These data suggest that CYT-0851 may be active as a mono therapy and a combinatorial therapy with PARP inhibitors in breast cancer. Overall, we conclude that the synergy observed in vitro indicates significant potential for RAD51 inhibition in future cancer treatment strategies and warrants future exploration in vivo. Citation Format: Joey L Guy, Tyler Maclay, Melinda Day, Monika L Burness, Kevin Mills. RAD51 inhibition using CYT-0851, shows anti-cancer activity in cellular models of breast cancer and acts synergistically with PARP inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-05.

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We leverage a “gain-of-function” synthetic lethality approach to selectively target cancers that ectopically express Activation Induced Cytidine Deaminase (AID). AID is a DNA-directed cytidine deaminase normally transiently expressed in maturing B-lymphocytes where it plays a critical role in somatic hypermutation and immunoglobulin class switching. However, AID is dysregulated and inordinately expressed in many cancers, where it acts as a DNA-damaging enzyme mutating widespread locations throughout the genome. This activity leads to the accumulation of point mutations and DNA breaks and promotes DNA replication stress. In the latter context, AID-expressing cells become critically dependent on the homologous recombination factor RAD51 to survive DNA damage-induced replication stress. We have developed a novel small-molecule inhibitor of RAD51, CYT-0851, that is preferentially cytotoxic to AID expressing cells. We have shown in vitro and in vivo that CYT-0851 is efficacious in AID-expressing lymphoma and leukemia mouse models. Genome profiling and gene expression studies have shown many solid tumors, including pancreatic cancer, also aberrantly express AID. To determine whether targeting RAD51 may be an effective approach to the treatment of AID-expressing pancreatic cancer, we evaluated the antitumor activity of CYT-0851 in multiple patient-derived pancreatic cancer xenografts (PDX) models with a range of AID expression levels. All models tested showed tumor growth inhibition (TGI), ranging from 63 to 104% with CYT-0851 treatment. A crossover experiment was performed with one model to determine the activity of CYT-0851 in large, established tumors. We observed tumor regression (137% TGI) in large tumors upon treatment with CYT-0851, resulting in one partial responder and one tumor-free responder. The data presented here demonstrate that CYT-0851 exerts anticancer activity in multiple preclinical models of pancreatic cancer. Considering that recent published studies have shown that these cancers can ectopically overexpress AID, we conclude that CYT-0851 may be an effective treatment for pancreatic cancer. Overall, this provides a rationale for the continued development of CYT-0851 as a novel therapeutic for pancreatic cancer patients. Citation Format: Melinda Day, Jean-Marc Lapierre, Tom O'Shea, Kevin Mills. A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C14.

Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

AID, a DNA-directed cytidine deaminase, plays a critical role in somatic hypermutation and immunoglobulin class switching in maturing B-lymphocytes. Unlike site specific recombinases such as RAG1/2, AID is a promiscuous DNA damaging enzyme that deaminates cytidines at sites throughout the genome. AID expressing cells become critically dependent on the homologous recombination factor RAD51 to repair DNA double strand breaks that result from these off target deamination events. We have developed a novel small molecule, CYT01B, which inhibits RAD51 response to DSBs and is potent to a much greater extent in AID expressing cells. Previously, we have shown this molecule to have activity in mouse lymphoma xenograft models that constitutively express AID. Here we present new preclinical characterization data of CYT01B in solid cancer models. We first analyzed the frequency and levels of AID overexpression in solid tumor data in The Cancer Genome Atlas. Multiple solid tumor types displayed ectopic AID expression, including breast cancer, sarcoma, melanoma, pancreatic cancer, lung cancer, and head and neck cancers with about 30% of the patient samples 4-fold above the baseline expression level. We then tested CYT01B in several solid tumor derived human cell lines. We observed a correlation between AID expression and activity. Cell lines with low ectopic expression of AID had EC50 values in the low micromolar range (~2µM), while those without AID expression gave EC50 values of about 5µM and higher. Next, we examined the activity of our small molecule in 14 different PDX models with various levels of AID expression. These models included renal, head and neck, lung, pancreatic, ovarian, colorectal, and breast cancer samples. We observed a wide range of tumor growth inhibition across the different models (0% to 60%+), which tended to correspond with AID expression; the higher the AID expression the greater the observed tumor growth inhibition. Taken together, these data demonstrate that CYT01B shows anti-cancer activity in a range of solid tumor models. Overall, this provides the basis for continued development of CYT01B as an AID/RAD51 synthetic lethal therapeutic for solid cancers. Citation Format: Melinda Day, Tyler Maclay, Kevin Mills. A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4730.

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We have developed a novel RAD51 inhibitor, CYT01B, which we have previously shown to be preferentially active in Activation Induced Cytidine Deaminase (AID) expressing cells, and in other tumor derived cell lines with elevated levels of DNA damage and/or reduced DNA repair capabilities. CYT01B acts by reducing RAD51 focus formation, and depleting the nuclear pool of RAD51. We have observed anti-tumor effects with CYT01B in preclinical models, but have yet to determine if RAD51 could act as a sensitizer to current therapeutics. A main resistance mechanism to PARP inhibitors is the overexpression of RAD51, making it an attractive potential combination for our RAD51 inhibitor. To determine potential drug synergy, a matrix study was performed with CYT01B (concentration range of 20nM to 5µM) and 5 different PARP inhibitors including olaparib (78nM to 5µM), niraparib (78nM to 5µM), veliparib (780nM to 50µM), rucaparib (156nM to 10µM), and talazoparib (39nM to 2.5µM). The combination matrix was tested in cell lines of varying AID expression and PARP inhibitor sensitivity: ARPE19/HPV16 (HPV immortalized epithelial cell line), KYSE-70 (head and neck cancer cell line), Daudi (Burkitt’s Lymphoma cell line), HCC1143 (TNBC), and BT20 (TNBC). We used both the Loewe Additivity model and the Bliss Independence model to determine drug interaction (synergistic, independent, or antagonistic). In general, we observed synergy with all the combinations in the tumor derived cell lines. In the transformed epithelial cell line, synergy was observed only in the olaparib combination. The strength of the synergistic effect differed amongst the PARP inhibitors, with olaparib giving the strongest synergy scores. From this data we hypothesize that the synergistic activity with CYT01B is related to the PARP trapping efficiency of the PARP inhibitors; greater trapping efficiency the greater the synergy. These data suggest that CYT01B may be active as a combinatorial therapy with PARP inhibitors. Overall, we conclude that there is significant potential for combining RAD51 and PARP inhibition in future cancer treatment strategies, and warrants future exploration in vivo. Citation Format: Tyler Maclay, Melinda Day, Kevin Mills. CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 363.