• Identification and Characterization of Regorafenib and NU7441 as Targeted Immunotherapies for Melanoma

      Tsai, Alexander; Davila, Eduardo, Ph.D. (2017)
      The emergence of new therapeutics, including targeted therapies and immunotherapies, has improved prognoses for melanoma patients. Yet, each class of therapy suffers from unique shortcomings. Targeted therapies are only approved for approximately half of melanoma patients that carry a certain mutation in the BRAF gene. Additionally, responses to targeted therapies are almost always transient, as resistance arises rapidly. Immunotherapies offer, for the first time, the potential for long-term responses and complete regressions. However, these remarkable responses only occur in a small fraction of patients. The use of combination therapies is therefore emerging as a popular strategy to further improve outcomes in melanoma patients. The challenge is to identify appropriate agents that can be rationally combined to improve patient responses. We hypothesized that a targeted therapeutic that also altered the immunoregulatory milieu in a tumor might best synergize with an immunotherapy. Toward this end, high-throughput flow cytometry-based screening was performed with two large and diverse drug libraries to identify candidate therapies that augment T cell immunotherapy efficacy. Two lead therapies, regorafenib and NU7441, were selected based on their ability to alter a variety of immunomodulatory proteins, including CD55, CD73, CD155, programmed death ligand 1 (PD-L1), nerve growth factor receptor (NGFR), and human leukocyte antigen (HLA) class I in a heterogeneous panel of melanomas. The therapies were also found to upregulate several melanoma antigens, inhibit proliferation, and perturb commonly activated oncogenic signaling pathways in melanomas. T cells treated with the therapies proliferated normally and expressed increased CD25, CD28, inducible T cell costimulator (ICOS), and reduced co-inhibitory receptors. These phenotypic changes were associated with improved T cell function. In murine models, the compounds suppressed melanoma progression and altered various T cell subsets in the tumor microenvironment when used with and without various immunotherapies. Finally, regorafenib skewed intratumoral macrophages away from a phenotype associated with pro-tumor function. Collectively, these studies demonstrate for the first time that regorafenib and NU7441 influence the expression of immunomodulatory proteins on both tumor cells and T cells, and enhance the efficacy of various immunotherapies.
    • Regulation of Alt-NHEJ Repair and Devising Novel Targeted Therapies Involving PARP1 in Triple Negative Breast Cancer

      Chowdhury, Khadiza; Rassool, Feyruz V. (2015)
      Triple negative breast cancers (TNBCs) are one of the most clinically challenging sub-types of breast cancers with high genomic complexity and heterogeneity making it difficult to devise targeted therapies against them. Deficiency in repair of potentially lethal DNA double strand breaks (DSBs), including deletions/mutations of BRCA1/2 homologous recombination (HR) repair genes are associated with acquisition of chromosomal aberrations and translocations that can lead to disease progression. Recent studies in TNBCs from the Rassool laboratory have reported elevated expressions of LIG3 and PARP1, components of highly error-prone alternative-non-homologous end-joining (Alt-NHEJ) pathway for repairing DSBs. Thus, deficient HR is thought to lead to compensatory repair of DSBs by Alt-NHEJ, likely leading to genomic instability. In addition, the Rassool Laboratory has reported that increased Alt-NHEJ may be a mechanism for survival in TNBCs. However, the mechanism through which TNBCs regulate Alt-NHEJ is not understood. Elevated levels of PARP1 make TNBCs potential therapeutic targets for PARP inhibitors (PARPi) that are known to catalytically inhibit DNA repair functions of PARP1 as well as trap PARP1 in chromatin, forming cytotoxic DNA-PARP1 complexes. However, clinical trials involving PARPis as single agents of treatment of BRCA-deficient or BRCA-proficient TNBCs have failed to demonstrate sustained responses, suggesting that PARPis may need to be combined with other therapies. In addition to exhibiting high levels of PARP1, our preliminary data demonstrate that TNBCs express increased levels of DNA methylation factor, DNA methyl transferase 1 (DNMT1). In addition, PARP1 has been reported to interact with DNMT1, and these above observations suggest that a combination treatment of PARPi and DNMTi might enhance anti-tumor responses in TNBCs. In this study we investigated in both BRCA -proficient and -deficient TNBCs: i) mechanism(s) underlying Alt-NHEJ regulation and ii) determined whether therapy using PARPi and DNMTi enhance anti-tumor effects, in vitro and in vivo, compared with administration of PARPis alone. The first part of our investigation led to the discovery that the oncogene C-MYC which is frequently upregulated in TNBCs, is a transcriptional regulator of Alt-NHEJ components, LIG3 and PARP1, resulting in upregulation of Alt-NHEJ activity in TNBCs. In the next part of our study, we devised a promising new strategy to improve the efficacy of PARPi when combined with DNMTi in TNBCs. Combination treatment showed significant reduction in clonogenicity and strong anti-tumor effects in BRCA -proficient and -deficient cell lines and mouse xenograft models. An initial insight into the mechanisms for this increased sensitivity of the drug combination revealed a significant increase in PARP1 trapping which correlates with increased levels of cytotoxic DSBs. Thus our study provides compelling pre-clinical results suggesting that TNBCs with elevated PARP1 and DNMT1 levels are potential targets for PARPi and DNMTi combination treatment. Since both drugs are in clinical use, these studies lay the groundwork for the development of clinical trials to treat these devastating diseases.