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Tumor stroma is critical in shaping the tumor microenvironment and promoting tumor growth and metastasis. Six stroma-targeted kinase inhibitors (cediranib, vandetanib, axitinib, pazopanib, cabozantinib, and lenvatinib) were tested both as single agents and in combination with five tumor cell-targeted kinase inhibitors (erlotinib, gefitinib, osimertinib, rociletinib, and erdafitinib)using a complex spheroid cell culture model. Complex spheroids, containing 60% tumor cells, 25% endothelial cells (HUVEC) and 15% mesenchymal stem cells (MSC), serve as a cell culture model of human solid tumors incorporating both malignant and stromal cells. Seventeen tumor cell lines were grown as complex spheroids, including varied sarcoma and non-small cell lung cancer types. Drug sensitivities of the HUVEC and MSC were also evaluated, both individually as monolayers and mixed as monolayers and spheroids. Overall, the HUVEC and MSC were more sensitive to the VEGFR inhibitors than to the EGFR inhibitors. Growth as complex spheroids reduced the sensitivity of HUVEC and MSC to several kinase inhibitors compared to each cell type alone or in mixed monolayer culture. A significant correlation was observed between the EGFR mRNA expression in patient-derived tumor lines and their sensitivity to the EGFR inhibitors. In combination regimens, Erlotinib was most effective when combined with the VEGFR inhibitors pazopanib, vandetanib and cediranib, which produced more than one log of cell killing at concentrations less than the clinical Cmax of each drug. Osimertinib and rociletinib, which irreversibly target EGFR variants, were more cytotoxic towards complex spheroids of NCI-H1975 NSCLC (EGFR L858R, T790M) than NCI-H522 NSCLC (EGFR wildtype). Combinations of the mutant-selective EGFR inhibitors with a VEGFR inhibitor (especially, vandetanib, lenvatinib or cediranib) increased the cytotoxicity in complex spheroids containing EGFR mutant and wildtype tumor lines. Highly effective combinations included erdafitinib with vandetanib and erlotinib with cediranib. The combination of cediranib and erlotinib was also evaluated in nine patient-derived xenograft (PDX) models and resulted in two partial responses in a penile squamous carcinoma and uterine sarcoma, while the remainder showed stable, slowed, or progressive disease. Using >1 log of cell kill at drug concentrations less than the mouse Cmax concentration for each drug as a predictor for in vivo responsiveness, the complex spheroid assay was 88% accurate in predicting response or progressive disease in the PDX models. This criteria for predicting in vivo activity from in vitro model results will be further explored. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Thomas S. Dexheimer, Julie Laudeman, Thomas Silvers, Rene Delosh, Russell Reinhart, Chad Ogle, Eric Jones, Nathan P. Coussens, Beverly A. Teicher, Naoko Takebe, Alice Chen, James H. Doroshow. Combinations of receptor tyrosine kinase inhibitors targeting the tumor and stromal cells of complex spheroids from the National Cancer Institute’s Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/) [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 P222.

Background: TRC102 binds to abasic sites in DNA, inhibiting the base excision repair (BER) pathway, which is implicated in resistance to alkylating agents. Preclinical xenograft studies in mice demonstrated a synergistic anti-tumor effect when TRC102 was combined with alkylating agents. TRC102 given in combination with temozolomide in a phase 1 trial demonstrated acceptable toxicity and antitumor activity in patients (pts) with colorectal cancer (CRC), non-small cell lung cancer (NSCLC, squamous cell carcinoma) and granulosa cell ovarian cancer (Oncotarget 2020). Pharmacodynamic (PD) analysis revealed treatment-induced induction of nuclear Rad51 signal, indicating DNA damage repair (DDR) response and tumor biopsy data indicated MGMT methylation correlated with tumor response in CRC (Cancer Cell, 2020). Here we report data from a phase II expansion cohort of this combination in pts with NSCLC. Methods: We conducted a single arm open label trial in a pt cohort with advanced NSCLS, who had received at least one line of therapy in the metastatic setting. Pts who received immunotherapy (ICI) and/or molecularly targeted therapy were eligible. Pts received 125 mg TRC102 (100 mg for BSA < 1.6) and 150 mg/m2 TMZ given orally daily for days 1-5 of 28-day cycle (C). Restaging CT scans were carried out after every 2 C and evaluated using RECIST 1.1. Blood from all pts was analyzed for circulating tumor cells (CTCs) at baseline and the beginning of each C. A two-stage design was employed, with at least 2 objective responses needed among the first 16 pts treated on study in order to proceed to the second stage. Results: Sixteen pts with NSCLC (12 adenocarcinoma, 4 squamous) were enrolled between 11/2016 and 6/2021; 12 men and 4 women, median age of 69 yrs (range 33-80), 1 Hispanic, 3 Asian, 2 Black/African American, and 10 Caucasian. Pts received a median of 3 prior lines of therapy (range 1-7), including ICI in all but 1 pt. Twelve pts were evaluable for response. Three pts experienced clinical progression before completing C 2, and 1 pt withdrew consent before completing C 2. Median number of Cs received by pts was 3 (range 1-14); 9 pts experience stable disease (SD) as best response lasting median of 12 wks (range 8-56 wks), and 4 pts experienced prolonged SD lasting greater than 6 Cs (24 wks). Treatment-related grade 3 adverse events reported include lymphopenia (4) and anemia (1). No grade 4 or 5 events were noted. Conclusion: TRC102 in combination with temozolomide was well tolerated with notable efficacy. Although no objective tumor responses were observed in this heavily pretreated population, 9 of 15 pts experienced SD, lasting ≥ 24 wks in 4 pts. Given the limited options for NSCLC treatment following ICI and TKI treatment, this regimen merits further evaluation. Analysis of CTCs is ongoing, as well as additional biomarker evaluation in other treatment cohorts. Funded by NCI Contract No. HHSN261200800001E Citation Format: Mohamad A. Salkeni, Geraldine O’Sullivan Coyne, Jennifer Zlott, Naoko Takebe, Ning Ma, Larry Rubinstein, Richard Piekarz, Jared Foster, Brandon Miller, Ralph Parchment, Abdul R. Naqash, Andrea Voth, James Doroshow, Alice Chen. Phase II trial of TRC102 (methoxyamine HCl) in combination with temozolomide (TMZ) in patients with advanced non-small cell lung cancer [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 P049.

Background: We initiated the systematic NCI-ALMANAC in vitro combination drug screen of FDA-approved anticancer drugs in the NCI-60 tumor cell line panel to explore novel and effective drug combinations (Holbeck et al., Cancer Res 2017). In this screen and in preclinical xenograft models, the BCR-ABL kinase inhibitor nilotinib demonstrated greater-than-additive activity in combination with the anti-tubulin agent paclitaxel. Additionally, recent preclinical findings suggest a reduced incidence of peripheral neuropathy for this combination relative to paclitaxel monotherapy (Leblanc et al., J Clin Invest 2018). We are performing a phase 1 study to establish the safety, tolerability, and maximum tolerated dose (MTD) of this combination in patients (pts) with advanced solid tumors, and to examine the pharmacokinetic (PK) and pharmacodynamic (PD) effects of the combination to better understand the mechanism of synergy. Materials and Methods: Nilotinib was given orally twice-daily (BID), while paclitaxel was administered intravenously on days 1, 8, and 15 of each 28-day cycle. A 1- or 2-day paclitaxel-only run-in during the first cycle enabled comparison of the PK and PD effects of the combination vs. single-agent paclitaxel. Dose-limiting toxicities (DLTs) were assessed during cycle 1, and response was evaluated by CT per RECIST 1.1. Results: Thirty pts have been enrolled to date. The MTD was 300 mg nilotinib BID and 80 mg/m2 paclitaxel given on days 1, 8, and 15; DLTs were grade 4 rash and grade 3 myelosuppression. Only 2 pts (7%) have experienced grade 2 peripheral neuropathy, no grade ≥3 neuropathy events occurred. Of the 24 pts assessable for response to date, there are 4 confirmed partial responses (PRs; 17%, 1 pt with endometrial cancer, 1pt with anal cancer and 2 pts with granulosa cell of the ovary) and 12 pts with a best response of stable disease (SD; 50%), including 6 pts with SD for ≥ 8 cycles. Mean time on study to date is 8.7 months (range: 2 – 41 months). Three of 4 responding pts had undergone prior paclitaxel-based therapy and experienced a best response of only SD (1 pt) or progressive disease (2 pts) to carboplatin/paclitaxel or paclitaxel monotherapy, respectively. PK data demonstrate dose-dependent plasma paclitaxel concentrations (for 60 vs. 80 mg/m2 paclitaxel), and no substantial effects of nilotinib on paclitaxel plasma concentrations. Conclusions: The combination of nilotinib and paclitaxel is well-tolerated. Consistent with recent preclinical findings, no grade ≥ 3 peripheral neuropathy has been observed, as compared to rates of 8-30% in prior studies of 80-100 mg/m2 weekly paclitaxel monotherapy in pts with metastatic breast cancer (Rivera and Cianfrocca, Cancer Chemother Pharmacol 2015). PK data indicate dose-dependent plasma paclitaxel concentrations, with no substantial nilotinib-induced changes in plasma paclitaxel levels. Preliminary response data are promising, especially considering the PRs observed in pts who had previously been unresponsive to paclitaxel therapy. Accrual to the expansion cohort and PD analyses of circulating tumor cells and tumor biopsy specimens are ongoing. Citation Format: Geraldine O'Sullivan Coyne, Shivaani Kummar, Jennifer Zlott, Lamin Juwara, Naoko Takebe, Murielle Hogu, Larry Anderson, Jerry Collins, Richard Piekarz, Howard Streicher, Elad Sharon, Arjun Mittra, Sabrina Khan, Brandon Miller, Tony Navas, Apurva Srivastava, Ralph Parchment, Laurence Rubinstein, James H Doroshow, Alice P Chen. Tolerability and antitumor activity of paclitaxel is improved by the addition of nilotinib in patients with refractory solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C008. doi:10.1158/1535-7163.TARG-19-C008

Background: The NCI ALMANAC study carried out a systemic in vitro drug screen with 2.8 million dose combinations to identify drug pairs with greater than additive activity. This was followed by patient-derived xenograft models, which demonstrated that the combination of the proteasome inhibitor bortezomib and the nucleoside analog clofarabine was highly active. Both agents have been approved by FDA: bortezomib for multiple myeloma & mantle cell lymphoma, and clofarabine for acute lymphoblastic leukemia. However, neither agent is approved for solid tumors or myelodysplastic syndrome (MDS). Our pre-clinical models suggest that the activity of this drug pair may be due to modulation of DNA damage and enhancement of the intrinsic apoptotic cascade. While both drugs can initiate apoptosis, bortezomib resistance occurs when the drug fails to upregulate the apoptotic promoter BAX; however, clofarabine can stabilize BAX and facilitate apoptosis via alternate pathways (unpublished data). We are conducting an ongoing phase 1 trial to evaluate the activity of bortezomib and clofarabine in refractory solid tumors, lymphomas, and MDS. Methods: The trial uses a 3+3 design, enrolling at least one solid tumor/lymphoma and one MDS patient (pt) at each dose level. The occurrence of pre-defined hematologic dose limiting toxicity (DLT) or a second instance of a grade 2 hematologic toxicity triggers expansion of the dose level into separate cohorts: (1) solid tumor/lymphoma & (2) MDS. Starting dose of clofarabine was 1 mg/m2 administered intravenously (IV) on day (D)1-5, with bortezomib 0.8 mg/m2 SQ on D1-4 of 21-day cycles. Response was measured using RECIST 1.1, Lugano criteria, and IWG, respectively. Exploratory endpoints include DNA damage and epithelial-to-mesenchymal phenotype transition in blood and malignant tissue. Results: As of July 2019, 15 pts were enrolled with advanced solid tumors (n=11), lymphoma (1) & MDS (3). Median patient age is 62 (48-80 yrs). Median lines of prior therapy is 3 (1-7). One pt had a DLT (grade 3 anemia in a solid tumor pt at dose level 3), leading to the separation into solid tumor/lymphoma and MDS cohorts. In the MDS cohort, 2 pts had stable disease (SD) as best response (1pt with >8 months duration of response [DOR] following hypomethylating agent [HMA] failure). In the solid tumor/lymphoma cohort, 2 pts achieved a best response of SD (1pt with > 7 months DOR, with colorectal adenocarcinoma). The only toxicity possibly attributed to treatment in the MDS cohort was grade 3 neutropenia. In the solid tumor/lymphoma cohort, the grade 3 toxicities were anemia (2) and lymphopenia (4); there was one grade 4 lymphopenia, and no grade 3/4 neutropenia or thrombocytopenia. Conclusions: This unique trial design demonstrates that conducting a phase 1 study with both solid tumors and hematologic malignancies is feasible and efficient, eliminating the need for a larger sample size or two separate trials. Treatment with bortezomib and clofarabine has shown early activity in a colon cancer pt and an MDS pt post HMA failure, where treatment options are critically limited. Dose escalation and pharmacodynamic studies are ongoing. Future studies include assessment of drug pair mechanism of action using a validated apoptosis multiplex assay and next-generation sequencing. Funded by NCI Contract No. HHSN261200800001E Citation Format: Sabrina S. Khan, Geraldine O'Sullivan, Larry Rubinstein, Shivaani Kummar, Lamin Juwara, Jennifer Zlott, Arjun Mittra, Murielle Hogu, Jerry Collins, Apurva Srivastava, Brandon Miller, Ralph E. Parchment, Christopher S. Hourigan, Steven Pavletic, James Doroshow, Alice P. Chen, Naoko Takebe. Phase I trial of the combination of bortezomib and clofarabine in adults with refractory solid tumors, lymphomas, or myelodysplastic syndromes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A071. doi:10.1158/1535-7163.TARG-19-A071

Background: The PI3K pathway is frequently dysregulated in cancer, promoting tumorigenesis by facilitating aberrant cell growth, blocking apoptotic signaling, and suppressing immune surveillance in the tumor microenvironment (TME). The TME contains regulatory immune cells such as FOXP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells, M2 macrophages, and N2 neutrophils, maintaining an immunologically isolating sheath. PI3K inhibition has been shown to slow tumor growth by inducing apoptosis and sensitizing tumors to T cell-mediated cytotoxicity. Copanlisib is a potent PI3K inhibitor, and combination with the PD-1 checkpoint inhibitor nivolumab is hypothesized to result in robust anti-tumor T cell responses. Materials and Methods: Open label phase I trial of copanlisib and nivolumab following a 3+3 design with an expansion phase at the maximally tolerated dose (MTD). Flat-dose nivolumab is administered intravenously (IV) on day 1 while copanlisib is administered IV weekly on days 1, 8 and 15 of a 28-day cycle; there is a 2-week copanlisib run-in during cycle 1 (expansion phase) to enable assessment of pharmacodynamic (PD) endpoints. Prior checkpoint inhibitor therapy is not permitted. Dose-limiting toxicities (DLTs) were assessed during cycle 1, and responses are assessed by CT using RECIST 1.1 and iRECIST. Exploratory PD objectives include assessments on blood and tissue samples collected at baseline and post copanlisib -/+ nivolumab treatment; these studies will evaluate immune cell populations, PD-1/PD-L1 levels, markers of T cell activation/inhibition (Zap70 pY493 and SHP2 pY542), and apoptosis biomarkers using validated immunofluorescence and flow cytometry assays. Results: As of July 2019, 16 pts have been enrolled with 6 pts in the expansion phase; 14 pts are evaluable for toxicity. The MTD for the combination is copanlisib 60 mg days 1, 8 and 15 plus nivolumab 480 mg day 1 (recommended doses for each individual agent, 28-day cycles); no DLTs were identified. Median age on study is 62 (range 38-77). One pt (rectal cancer) has experienced a confirmed partial response (PR), and 4 pts have achieved stable disease (SD; 1 pt remains on study ≥10 months). Five pts are too early to assess for response. Tissue for molecular analysis and/or genomic sequencing has been collected for all patients. Adverse events include: grade 3 hypertension (1 pt, requiring dose reduction) and infusion-related reaction (hypertension, 1pt); grade 1/2 events include hypertension (7 pts), hyperglycemia (5 pts), nausea (5 pts) and infusion-related reaction (hypertension, 4 pts). Preliminary PD assessments show increases in the number of CD8+ T cells in peripheral blood samples after treatment, with changes in phospho-biomarkers of T cell activation (Zap70-pY493 and SHP-pY542). Conclusions: The combination is tolerable using the approved doses of each individual agent and shows treatment-induced immune PD changes as well as early anti-tumor activity. Enrollment at the expansion stage is ongoing (NCT03502733). Funded by NCI Contract No. HHSN261200800001E. Citation Format: Geraldine O'Sullivan Coyne, Timothy A Yap, Nancy Moore, Arjun Mittra, Naoko Takebe, Lamin Juwara, Sabrina Khan, Ecaterina Ileana Dumbrava, Howard Streicher, Richard Piekarz, Elad Sharon, Funda Meric-Bernstam, Ralph Parchment, Tony Navas, King Leung Fung, James Mitchell, Kristin Fino, Sandra Montez, Austin Doyle, Laurence Rubinstein, James H Doroshow, Alice P Chen. Phase IB combination study of copanlisib and nivolumab in advanced solid tumors and lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B105. doi:10.1158/1535-7163.TARG-19-B105

Background. In vitro experiments from multiple laboratories have shown that the methylation-induced silencing of the p16INK4A gene, a tumor suppressor gene, can be reversed by DNA methyltransferase (DNMT1) inhibitor-based epigenetic modulation. However, it remains unclear whether DNMT1 inhibitors induce re-expression of p16INK4A in patients treated with demethylation promoting agents. Here we report p16INK4A expression status in human circulating tumor cells (CTCs) and its dynamic changes during DNMT1 inhibitor therapy in patients with advanced solid tumors, by using a novel CTC-based p16INK4A immunofluorescence assay. Methods. We demonstrated in vitro re-expression of p16 by Western blot and whole cell immunofluorescence in EJ-6 cells treated with decitabine, 4-thio-2’-deoxycytidine (TdCyd) and 5-fluoro-2’-deoxyctidine (FdC) but not in the p16-deleted line A549. We analyzed p16 re-expression in epithelial /mesenchymal CTC subpopulations as a clinical pharmacodynamic (PD) marker to assess the effects of DNMT1 inhibitors (analyzed using CellSearch® and fluorescence microscopy). Results. Over 300 specimens from 70 patients were analyzed from patients enrolled in both Phase 1 and Phase 2 clinical trials of the DNMT1 inhibitors TdCyd or FdC in combination with tetrahydrouridine (THU). Circulating tumor cells were characterized as DAPI+/CD45-/CEA or MUC1 positive, then classified as epithelial or mesenchymal based on expression of (pan-)cytokeratin (CK) or vimentin (VIM), respectively. The p16INK4A-positive CTC baseline level ranged from 0 to 15% in 57 of 58 patients with assessable CK+CTC numbers and in 46 of 53 patients with assessable VIM+CTC numbers. There were no patient specimens with CTCs that were positive for only VIM, but most specimens had higher numbers of VIM+ CTC compared to CK+ CTC, and not all patients had both CTC phenotypes present at baseline. During treatment with DNMT1 inhibitors, p16INK4A-positive CTCs increased in 46 of 58 patients with assessable CK+ CTCs (epithelial phenotype), but only 6 patients with VIM+ CTCs (mesenchymal phenotype) had p16 re-expression, and these patients had both CTC phenotypes present. The restoration of p16INK4A expression in CTC, not the CTC number, was correlated with evidence of drug clinical activity in patients. Drug treatment longer than 2 cycles was associated with both EMT phenotypic conversion and obvious morphological changes in CTCs that accompanied increased p16INK4A expression. Conclusions. Our studies indicate that monitoring dynamic changes of CTC-based p16INK4A expression is a sensitive PD biomarker for assessing DNMT1-targeted epigenetic anticancer therapeutics in clinical development. CTCs were predominantly mesenchymal phenotype. In addition, the CTC phenotype (epithelial vs mesenchymal) appeared to be quite dynamic in treated patients. Funded by NCI Contract No. HHSN261200800001E, and Cooperative Agreements U01CA062505 and UM1CA186717. Citation Format: Lihua Wang, Brandon Miller, Sonny Khin, Geraldine O’Sullivan-Coyne, Alice P. Chen, Shivaani Kummar, Naoko Takebe, Kate Ferry-Galow, Ralph E. Parchment, Larry Rubinstein, Richard Piekarz, Edward M. Newman, James H. Doroshow, Robert J. Kinders. Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B018. doi:10.1158/1535-7163.TARG-19-B018

Background: Modulation of BRCA1 and ATR has been postulated as an activation mechanism of checkpoint 1 (Chk1), which in turn triggers homologous recombination repair through modulation of BRCA2-Rad51, and the initiation of cell cycle checkpoints through phosphorylation and activation of Wee1. Wee1 is a tyrosine kinase implicated in the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1/CDC2)-bound cyclin B, resulting in G2 cell cycle arrest in response to DNA damage. AZD1775 is a first-in-class selective inhibitor of Wee1 kinase that directly prevents phosphorylation of CDC2 at Tyr15. Model data show single-agent antitumor activity in multiple cancer cell lines across the NCI-60 and in both p53 intact and deficient tumor xenografts. We are conducting a phase I trial of AZD1775 in patients (pts) with refractory solid tumors, both with and without germline BRCA1/2 mutations (BRCA+), evaluating two different dosing schedules. We have previously reported on initial antitumor activity and proof-of-mechanism (Do et al, J Clin Oncol. 2015). This abstract reports the efficacy seen in the BRCA+ population. Methods: AZD1775 was administered orally twice daily (BID) for 5 doses [D1-3 and 8-10, Cohort A (A)] or once daily for 5 days [D1-5 and 8-12, Cohort B (B)] during weeks 1 and 2 of a 21-day cycle; 3+3 design. Primary objectives: establish safety, tolerability and PK of AZD1775. Secondary objectives: determine the effect of AZD1775 on markers of DNA damage and apoptosis in tumor tissue/circulating tumor cells, and antitumor activity. Dose-limiting toxicity (DLT) was determined during Cycle 1. Response defined by RECIST 1.1 on CT. Results: A total of 17 BRCA+ pts have enrolled; 9 pts on A (one pt withdrew for other therapy), and 8 pts on B. Nine pts with BRCA1, 7 pts with BRCA2: ovarian (9 pts), breast (2), pancreas (1), prostate (1), Fallopian tube (1), and peritoneal (1) cancer. Partial responses (PR) were confirmed on 2/8 pts (25%) in A with BRCA1 papillary serous ovarian cancer and squamous cell carcinoma of tongue. Two additional PRs (25%) were confirmed in BRCA1 serous ovarian cancer pts in B. Two further BRCA1 ovarian pts trended near PR. All 4 responders received prior platinum therapy; 3/3 ovarian cancer pts received and responded to prior PARP inhibitor therapy. No differences were noted in severity or rate of toxicity in pts with/without BRCA+: common grade 3/4 toxicities include anemia (6 pts, 37%), lymphopenia (5, 31%), neutropenia (4, 25%), and thrombocytopenia (3, 18%), occurring in responders and nonresponders. Conclusions: AZD1775 has single-agent antitumor activity in a BRCA deficient population, especially BRCA1. Responses were observed in pts with prior exposure to platinum and a PARP inhibitor. Additional accrual on a safety expansion cohort is ongoing, and assessment of PD biomarkers is planned for paired biopsies (NCT01748825). Citation Format: Geraldine OSullivan Coyne, Shivaani Kummar, Naoko Takebe, Khanh Tu Do, Robert Meehan, Richard Piekarz, Jennifer Zlott, Lamin Juwara, Mary Quinn, Elad Sharon, Howard Streicher, Lawrence Rubinstein, Chana Levine, James H. Doroshow, Alice P. Chen. Single agent AZD 1775, a Wee1 inhibitor, shows activity in BRCA deficient patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B079.

Multiple myeloma is an incurable disease for the majority of patients, therefore requiring new biological targeted therapies. In primary myeloma cells, IMP dehydrogenase (IMPDH) was shown to be consistently overexpressed. We therefore tested the IMPDH inhibitor mycophenolate mofetil (MMF) currently available as a clinical therapeutic agent for its antimyeloma activity in vitro. MMF depleted intracellular guanosine 5′-triphosphate (GTP) levels in myeloma cells. We showed apoptosis induction in myeloma cell lines and primary myeloma cells between 1 and 5 μmol/L MMF. MMF was also cytotoxic at this concentration in dexamethasone-resistant and Mcl-1-overexpressed myeloma cell lines shown by the tetrazolium salt XTT assay along with cell survival measured by a modified flow cytometric assay. Apoptosis was not inhibited by the presence of an antioxidant, suggesting that MMF-induced apoptosis is less likely to be associated with reactive oxygen species. However, apoptosis was abrogated by exogenously added guanosine, which activates an alternative pathway for GTP formation, implicating that this effect is directly mediated by IMPDH inhibition. MMF-induced G1-S phase cell cycle arrest and its apoptosis induction mechanism were associated with a caspase-dependent pathway as shown by alteration of mitochondrial membrane potential and cytochrome c release followed by activation of the caspases. MMF-induced apoptosis was also inhibited by a pan-caspase inhibitor Z-VAD-fmk. MMF-treated myeloma cells showed an up-regulation of Bak, which most likely together with Bax resulted in the release of cytochrome c. In summary, MMF attenuates G1-S phase cell cycle progression and activates the pathway of mitochondrial dysfunction, leading to cytochrome c release followed by activation of caspases. [Mol Cancer Ther 2006;5(2):457–66]

Cancer therapy strategies that selectively target tumors while sparing normal tissues would provide enormous clinical benefit to cancer patients. Central to developing these strategies is identification of tumor specific vulnerabilities and molecular targeted agents targeting these vulnerabilities. GMX1777/1778, a novel inhibitor of NAD+ biosynthesis, is a potent and specific inhibitor of the nicotinamide adnine dinucleotide (NAD+) biosynthesis enzyme, phosphoribosyl transferase (NAMPRT). Since cancer cells have a high rate of NAD+ turnover, modulation of NAD+ biosynthesis would be an attractive target for GMX 1777/1778. In addition to the depletion of NAD+ level, we present evidence of inducing other metabolic and oxidative stresses in a tumor specific manner with minimal damage to normal tissues in vitro and in vivo. Taken together, the cancer therapeutic strategy presented here would provide a novel way of specific killing of tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B98.