Mahrukh K. Ganapathi

Pharmacogenomics: New paradigms for targeted therapy based on individual response to drugs

Targeted therapy is the “holy grail” sought to achieve efficacy and reduce toxicity in cancer treatment. It is difficult nowadays to avoid reference to the word “targeted” or “personalized” in publications describing “novel” strategies for treating cancer. During the last decade, spectacular advances in genomics have brought the realization of targeted therapy closer to the bedside in treating patients with hematologic and solid tumors. Remarkable progress based on our understanding of aberrant genes and signaling has led to novel strategies for targeted therapy, understanding efficacy in select patients, and possible mechanisms leading to development of resistance. However, the absence of durable responses, rapid development of resistance to treatment, and dose limiting toxicity continue to be major hurdles that hamper progress. The focus of this seminar in Urologic Oncology is certainly timely given the availability of drugs and pathways that can be targeted in the treatment of urologic malignancies. To accompany this article, we have 2 excellent essays that provide a comprehensive and informative review on the current status and the future of targeted therapy in bladder cancer and renal cancer. Although it is unfortunate that we do not have an essay on prostate cancer where targeted therapy has led to remarkable progress in the clinical management of metastatic disease, in this editorial, the theme of targeted therapy in prostate cancer is addressed because evolving paradigms with this disease are relevant in understanding why we have successes or failures. Although chemotherapy with classical cytotoxic drugs does not often directly address putative drug targets that govern efficacy or toxicity in therapy, targeting of specific aberrant genes/pathways with small molecule inhibitors in cancer represents a more rational approach. However, similar to chemotherapy with cytotoxic drugs, presence of a presumed target per se does not necessarily assure efficacy, and we face similar problems with several of the small molecule–targeted drugs that are active clinically in select malignancies. Moreover, in this era of targeted therapy, the age-old problem of “intrinsic” and “acquired resistance” is an issue that continues to be poorly understood. Drugs that target specific mutations can be aided with a companion test to achieve selectivity, but this luxury does not exist with aberrant signaling pathways, e.g., angio

Mechanisms regulating resistance to inhibitors of topoisomerase II

Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIβ isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIβ is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.

HOXA4/HOXB3 gene expression signature as a biomarker of recurrence in patients with high-grade serous ovarian cancer following primary cytoreductive surgery and first-line adjuvant chemotherapy

OBJECTIVES Aberrant homeobox (HOX) gene expression is reported in high-grade serous ovarian carcinoma (HGSOC), however, its prognostic significance remains unclear. METHODS HOX genes associated with progression-free survival (PFS) in a discovery cohort of primary HGSOC samples with RNA sequencing data, and those previously reported to be associated with clinical outcomes, were selected for qPCR testing in an independent training cohort of primary HGSOC samples (n=71). A prognostic model for PFS was developed using univariate and multivariate Cox regression. Patients were stratified into risk groups that optimized the test statistic. The model was tested in an independent HGSOC cohort from The Cancer Genome Atlas (TCGA) (n=320). The effect of selected HOX genes on drug sensitivity and reactive oxygen species (ROS) accumulation was examined in vitro. RESULTS Of 23 HOX genes tested in the training cohort, HOXA4 (HR=1.20, 95% CI=1.07-1.34, P=0.002) and HOXB3 (HR=1.09, 95% CI=1.01-1.17, P=0.027) overexpression were significantly associated with shorter PFS in multivariate analysis. Based on the optimal cutoff of the HOXA4/HOXB3 risk score, median PFS was 16.9months (95% CI=14.6-21.2months) and not reached (>80months) for patients with high and low risk scores, respectively (HR=8.89, 95% CI=2.09-37.74, P<0.001). In TCGA, the HOXA4/HOXB3 risk score was significantly associated with disease-free survival (HR=1.44, 95% CI=1.00-2.09, P=0.048). HOXA4 or HOXB3 overexpression in ovarian cancer cells decreased sensitivity to cisplatin and attenuated the generation of cisplatin-induced ROS (P<0.05). CONCLUSIONS HOXA4/HOXB3 gene expression-based risk score may be useful for prognostic risk stratification and warrants prospective validation in HGSOC patients.

Expression profile of COL2A1 and the pseudogene SLC6A10P predicts tumor recurrence in high‐grade serous ovarian cancer

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high‐grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA‐Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time‐to‐recurrence (TTR). Kaplan–Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27–0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30–0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082–0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.

Biological role and clinical implications of homeobox genes in serous epithelial ovarian cancer

Homeobox (HOX) genes are a family of transcription factors that are essential regulators of development. HOX genes play important roles in normal reproductive physiology, as well as in the development and progression of serous carcinomas, the predominant and most aggressive subtype of epithelial ovarian cancer (EOC). This review discusses aberrant HOX gene expression in serous EOC and its impact on tumor development and progression. Further identification of HOX target genes may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of patients with serous EOC.

Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIβ in regulation of the decatenation checkpoint

Abstract Topoisomerase (topo) IIα and IIβ maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIβ. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIβ. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIβ, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIβ inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIβ function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIβ cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Clonal lineage of high grade serous ovarian cancer in a patient with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene encoding neurofibromin, which negatively regulates Ras signaling. NF1 patients have an increased risk of developing early onset breast cancer, however, the association between NF1 and high grade serous ovarian cancer (HGSOC) is unclear. Since most NF1-related tumors exhibit early biallelic inactivation of NF1, we evaluated the evolution of genetic alterations in HGSOC in an NF1 patient. Somatic variation analysis of whole exome sequencing of tumor samples from both ovaries and a peritoneal metastasis showed a clonal lineage originating from an ancestral clone within the left adnexa, which exhibited copy number (CN) loss of heterozygosity (LOH) in the region of chromosome 17 containing TP53, NF1, and BRCA1 and mutation of the other TP53 allele. This event led to biallelic inactivation of NF1 and TP53 and LOH for the BRCA1 germline mutation. Subsequent CN alterations were found in the dominant tumor clone in the left ovary and nearly 100% of tumor at other sites. Neurofibromin modeling studies suggested that the germline NF1 mutation could potentially alter protein function. These results demonstrate early, biallelic inactivation of neurofibromin in HGSOC and highlight the potential of targeting RAS signaling in NF1 patients.

Characterisation of homologous recombination deficiency in paired primary and recurrent high-grade serous ovarian cancer

BackgroundHomologous recombination deficiency (HRD) is shown to predict response to DNA-damaging therapies in patients with high-grade serous ovarian cancer (HGSOC); however, changes in HRD during progression remains unknown.MethodsHRD scores were evaluated in paired primary and/or recurrent HGSOC samples (N = 107) from 54 patients with adjuvant platinum-based chemotherapy. BRCA1/2 mutation, BRCA1 methylation, loss of heterozygosity (LOH), and HRD scores were characterised using tumour DNA-based next-generation sequencing assays.ResultsAmong 50 evaluable pairs (N = 100 samples), high intra-patient correlation in HRD score was observed (r2 = 0.93). BRCA1/2 mutations, BRCA1/2 LOH, and HRD were maintained between primary and recurrent samples, except for one pair in which a BRCA1 reversion mutation was identified in the recurrent sample. Despite the reversion, both samples were classified as having high HRD scores ( ≥ 42). All samples with BRCA1/2 mutations exhibited high HRD scores; however, high HRD scores were more prevalent than BRCA1/2 mutations (55% vs. 30%, respectively).ConclusionMarkers of HRD were maintained between the primary and recurrent samples, regardless of other genomic changes that occurred during recurrence. HRD score/markers in primary tumours may be valuable and adequate for selection of platinum-based therapy and/or poly-ADP-ribose-polymerase (PARP) inhibitors in recurrent HGSOC.

Abstract 1186: Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway

Pharmacologic inhibition or molecular down-regulation of topoisomerase 2β (TOP2β) potentiates all trans retinoic acid (ATRA)-induced differentiation and apoptosis in acute myeloid leukemia (AML) cells. Since ATRA-induced myeloid differentiation involves activation of the MAPK/ERK signaling pathway and aberrant activation of MAPK/ERK is frequently observed in AML we tested the role of this pathway in modulating the cellular effects of ATRA when TOP2β is inhibited with the bisdioxopiperazines, ICRF 187 (dexrazoxane, Zinecard™) or ICRF193. Treatment of HL-60 AML (M2) cells with ATRA increased the percentage of differentiated cells, as assessed by the nitroblue tetrazolium reduction assay, and enhanced levels of phosphorylated ERK. Paradoxically, while combination treatment with ATRA/ICRF187 enhanced differentiation (P Citation Format: Eric J. Norris, Amy Dorszynski, Aaron Lucander, Darla Destephanis, Ram Ganapathi, Mahrukh Ganapathi. Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1186.

Abstract 4828: Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma cell lines

As a single agent, melphalan is used as a preparative treatment prior to autologous stem cell transplant (ASCT) in patients with multiple myeloma (MM) given its myelosuppressive and anti-myeloma effects. While most patients achieve complete remission following ASCT, a majority will develop recurrent disease. Therefore, novel combinations that improve the anti-myeloma activity of melphalan may improve post-transplant outcomes. The first-in-class agent Filanesib (Fil) is a kinesin spindle protein inhibitor which induces mitotic arrest/apoptosis in proliferating cells and has demonstrated anti-myeloma activity in vitro and in vivo. In this study, we tested the effects of combination treatment with filanesib and melphalan in the human myeloma cell lines (HMCLs), U266, MM1S and NCI-H929. As single agents, melphalan and filanesib both inhibit myeloma cell proliferation as assessed by the MTS assay. However, when HMCLs were treated simultaneously with both drugs an antagonistic interaction was observed. This antagonism was in part due to cell cycle arrest in the S-phase, which prevented filanesib from downregulating the anti-apoptotic proteins, BCL-2 and MCL-1 and exerting its cytotoxic effect in the G2/M-phase.. To elucidate whether sequential treatment could enhance cytotoxicity, HMCLs were treated with either melphalan (1 hour) followed by filanesib (MelFil) for 48 hours or filanesib for 48 hours followed by 1 hour melphalan treatment (FilMel). Compared to the apoptosis observed with individual drugs, the MelFil sequence led to significantly lower apoptosis, whereas the the FilMel treatment led to enhanced apoptosis following 48 hours after drug washout. Furthermore examination of recovery of cell proliferation following seven days of recovery after drug removal revealed an increase in cell proliferation for the MelFil treatment compared to a decline in the number of viable cells for FilMel treatment. Taken together, our findings suggest that filanesib pretreatment prior to melphalan may be a clinically relevant therapeutic combination strategy. Moreover, our data underscore the importance of testing sequencing strategies for optimizing therapeutic efficacy of combination treatment. Citation Format: Darla Destephanis, Eric J. Norris, Brian Turnquist, Saad Usmani, Mahrukh Ganapathi, Ram Ganapathi. Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4828.