• Antagonism of the Alpha-Helix Mediated Protein-Protein Interactions of the Bcl-2 and c-Myc Oncoprotein Families: Proteomimetic and Small-molecule Strategies

      Yap, Jeremy; Fletcher, Steven (2014)
      The Bcl-2 oncoprotein family includes both anti- and pro-apoptotic proteins that are normally localized within the mitochondrial outer membrane. The over-expression of the anti-apoptotic proteins (such as Bcl-xL, Bcl-2, and Mcl-1) is associated with cancer and chemotherapeutic resistance. Pro-apoptotic Bcl-2 proteins (such as Bak and Bim) initiate the intrinsic apoptotic pathway via oligomerization at the mitochondrial membrane. However, in the presence of over-expressed anti-apoptotic Bcl-2 proteins, pro-apoptotic Bcl-2 proteins are sequestered and the intrinsic apoptotic pathway is antagonized. Specifically, the conserved BH3 alpha-helix of the pro-apoptotic proteins engage the hydrophobic binding crevices of the anti-apoptotic proteins largely through hydrophobic (i), (i + 3/4) and (i + 7) residues on one face of the helix. Though potent inhibitors of Bcl-2 and Bcl-xL have been identified, chemically diverse pan-Bcl-2 and Mcl-1 specific inhibitors are lacking. Inspired by the recent advances in alpha-helix mimicry and fragment-based drug design, we have successfully synthesized potent (Ki ~ 150 nM) pan-Bcl-2 inhibitors based on trisbenzamide and salicylate scaffolds and validated their activities in vitro. The c-Myc oncoprotein is an intrinsically disordered (ID) transcription factor of a vast number of genes that are involved in cell proliferation and growth. Similar to anti-apoptotic Bcl-2 proteins, overexpression of c-Myc is associated with a myriad of cancers such as prostate, breast, and lung tumors. Though biologically inactive in its ID monomeric form, the transcriptional activation of c-Myc is initiated upon binding its obligatory protein partner Max. The transcriptionally active c-Myc-Max heterodimers recognize and bind the hexanucleotide sequence 5'-CACGTG-3' on dsDNA, where the transactivation domain of c-Myc recruits additional transcriptional machinery. Owing to its ID properties, in the absence of Max, c-Myc does not exhibit any secondary structure that may function as a basis for drug design. While several c-Myc specific inhibitors have been identified through high-throughput screening, few structure-activity relationship (SAR) studies have been reported. Towards developing potent c-Myc inhibitors, we conducted an SAR study on the c-Myc inhibitor 10074-G5 (IC50 = 146 uM), which resulted in the discovery of an improved inhibitor, JY-3-094 (IC50 = 33 uM) whose ester prodrugs exhibited potent cell activities (IC50 < 10 uM).
    • 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.
    • The Role of c-MYC in the Regulation of Double-Strand Break Repair in Tyrosine Kinase Activated Leukemias

      Muvarak, Nidal Emilio; Rassool, Feyruz V. (2012)
      Leukemias expressing activated tyrosine kinases (TKs) BCR/ABL and FLT3/ITD activate signaling pathways that lead to increased survival and proliferation. Expression of these oncogenes also results in increased genomic instability, evidenced by altered double-strand break (DSB) repair that may result in increased genomic changes, leading to disease progression and resistance to therapy. There are two main pathways for DSB repair: the error-free homologous recombination (HR) pathway and the error-prone non-homologous end-joining (NHEJ) pathway. In BCR/ABL- and FLT3/ITD-positive leukemias, HR is characterized by increased single base-pair s and NHEJ shifts to a highly error-prone alternative pathway. Increased expression of transcripts encoded by key gene components of these repair mechanisms and translated proteins are involved in generating increased errors. Increased expression of HR component RAD51 leads to unfaithful repair. For NHEJ, components of the classical pathway (C-NHEJ) are decreased, and in turn, levels of ALT-NHEJ factor DNA ligase IIIα (LIG3) are increased, resulting in increased frequency of large DNA deletions. Evidence suggests that c-MYC is a good candidate for transcriptional regulation of both RAD51 and LIG3 in TK-activated leukemias: Expression of c-MYC is increased in TK-activated leukemias, putative binding sites for c-MYC exist in the promoters of these genes, and c-MYC regulates RAD51 expression in prostate cancer. In this study we tested the hypothesis that c-MYC plays a role in the transcriptional regulation and/or activity of RAD51 and LIG3. We demonstrated that (1) chemical and siRNA inhibition of c-MYC results in downregulation of LIG3 and RAD51 in FLT3/ITD- and BCR/ABL-positive cells; (2) downregulation of RAD51, but not LIG3, is attributed to c-MYC-mediated block of entry into S-phase; (3) c-MYC binds to the promoter of LIG3, and (4) importantly, c-MYC downregulation results in functional consequences for both HR and NHEJ: RAD51 foci formation is inhibited following exposure to ionizing radiation, and decreased LIG3 results in increased NHEJ repair fidelity. Based on these findings, we conclude that c-MYC plays an important role in the induction of genomic instability through regulation of double-strand break repair pathways that lead to increased errors. These results merit further exploration of mechanisms through which c-MYC functions in this process.