Lower MYC Research Articles

Abstract 2009: Evaluation of RNA-sequencing signatures with response to pembrolizumab (pembro) monotherapy in ipilimumab-naive patients (pts) with melanoma

Abstract Introduction: KEYNOTE-001 (NCT01295827) was a phase 1 study of pembro in pts with locally advanced or metastatic melanoma (parts B and D). KEYNOTE-006 (NCT01866319) was a randomized phase 3 study of pembro vs ipilimumab in pts with advanced melanoma. We explored potential relationships between key tumor microenvironment canonical signatures beyond T cell-inflamed gene expression profile (GEP) and response/resistance to pembro monotherapy in pts with melanoma. Methods: In pembro-treated, ipilimumab-naive pts from KEYNOTE-001 and KEYNOTE-006 with available RNA-sequencing and clinical data (n = 476), we analyzed association of 9 non-T cell-inflamed GEP canonical signatures (angiogenesis, gMDSC, glycolysis, hypoxia, Myc, proliferation, RAS, stromal/EMT/TGFβ, WNT) with clinical outcomes. Signatures were defined with TCGA and Moffitt databases (independent of any pembro trial) using a correlation algorithm to select individual genes for membership in the canonical signature based on correlation with reference signatures identified in literature or internal work. Relationships between RNA signatures and clinical outcomes were assessed using 2 models: 1 adjusted for ECOG PS and batch effect and 1 adjusted for T cell-inflamed GEP to understand the additional explanatory value of a non-T cell-inflamed GEP signature for clinical outcomes. P values were adjusted for multiplicity; significance was predefined at α = 0.05. Database cutoffs for clinical data: 11/27/2017 (KEYNOTE-001), 12/4/2017 (KEYNOTE-006). Results: T cell-inflamed GEP was statistically significantly associated with response to pembro (P < 0.0001). No canonical signatures showed statistically significant association with response/resistance to pembro after multiplicity adjustment when adjusting for T cell-inflamed GEP, ECOG PS, and batch effect. Without adjusting for T cell-inflamed GEP, Myc signature was the only signature identified as statistically significantly associated with response to pembro in the postulated direction of negative association. Prolonged OS, but not PFS, was observed in pts with a lower Myc signature. Myc signature did not show independent predictive value beyond T cell-inflamed GEP, indicating that interferon γ-related T-cell inflammation has a more dominant impact on immunotherapy efficacy than the Myc signature and other gene sets in immune-suppressive axes. Conclusions: In this exploratory analysis, no canonical signatures showed statistical significance after adjusting for T cell-inflamed GEP. Although observed only without adjusting for T cell-inflamed GEP, Myc signature was negatively associated with response in ipilimumab-naive pts with melanoma receiving pembro monotherapy. Citation Format: Adil Daud, Matteo S. Carlino, Antoni Ribas, Ana Arance, Jean-Jacques Grob, James Larkin, Georgina V. Long, Jacob Schachter, Andrea L. Webber, Clemens Krepler, Chunsheng Zhang, Jared Lunceford, Razvan Cristescu, Qing Zhao, Junshui Ma, Caroline Robert. Evaluation of RNA-sequencing signatures with response to pembrolizumab (pembro) monotherapy in ipilimumab-naive patients (pts) with melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2009.

Abstract 6433: Dual targeting MYC G-quadruplex and topoisomerase I: Lead discovery indenoisoquinolines for cancer therapy

Abstract G-quadruplexes are noncanonical four-stranded DNA or RNA secondary structures that have emerged as novel therapeutic targets. MYC is one of the most important oncogenes and is overexpressed in a number of human cancers. The G-quadruplex formed in the MYC oncogene promoter (MycG4) is a transcriptional silencer and amenable to small molecule targeting. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties as compared to the clinically used camptothecin anticancer drugs. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776), and LMP744, have entered phase I clinical trials in adults with relapsed solid tumors and lymphomas. However, some indenoisoquinolines showed potent anticancer activity without being strong topoisomerase I inhibition activity, suggesting additional mechanisms of action. Herein, we demonstrate that a large number of anticancer indenoisoquinolines strongly bind and stabilize MycG4 in vitro, using fluorescence, nuclear magnetic resonance (NMR), and circular dichroism (CD) spectroscopy, as well as gel electromobility shift assay (EMSA). In addition, MycG4-interactive indenoisoquinolines lower MYC mRNA and protein levels in vivo, as shown by western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments. These results indicate that targeting the MYC promoter G4 to downregulate MYC is a likely mechanism of action for the anticancer activities of these particular indenoisoquinolines. In addition, structure-activity-relationships of MycG4 recognition by indenoisoquinolines were investigated and elucidated the essential structural requirements for strong binding. The analysis of indenoisoquinoline derivatives for their MYC inhibitory, topoisomerase I inhibitory, and anticancer activity led to the discovery of a synergistic effect of MYC inhibition and topoisomerase I inhibition on anticancer activity. In conclusion, our result reveals MYC downregulation by MycG4 binding as a new mechanism of action for anticancer indenoisoquinolines. Moreover, we suggest dual targeting of MycG4 and topoisomerase I as a novel strategy for cancer therapy. Citation Format: Kaibo Wang. Dual targeting MYC G-quadruplex and topoisomerase I: Lead discovery indenoisoquinolines for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6433.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Quantitative Analysis of the Effects of MYC Overexpression on the Mutation Rate in Human Cells Using the PIG-A Gene

Abstract 4406It has been hypothesized that the early acquisition of genomic instability is essential to account for the high number of mutations commonly identified in human cancers. Previous reports have demonstrated that MYC overexpression results in gene amplification. Given the importance of MYC in Burkitt's lymphoma, we investigated the effects of MYC on spontaneous inactivating somatic mutations, using an assay that we have developed, based on the PIG-A gene. PIG-A is X-linked, thus a single mutation can disrupt its function. PIG-A encodes an enzyme required for the biosynthesis of glycosylphosphatidylinositol (GPI), which is required for the expression of a set of membrane proteins on the cell surface (e.g., CD48, CD52, CD55, and CD59). Expansion of PIG-A mutant stem cells occurs in PNH, and it is known from this disorder that a very broad spectrum of mutations can inactive PIG-A. Using PIG-A, we have previously determined the mutation rate (μ) in EBV transformed B-lymphoblastoid cell lines (BLCLs) from normal human donors, and we have demonstrated genomic instability in cell lines derived from hematologic malignancies. Here we have measured μ in P493, a cell line derived from normal human B cells, which overexpress MYC (normalized levels 17 × higher than BLCLs). P493 can also be induced to express lower levels of MYC, resulting in a lower growth rate, using tetracycline and estradiol. To measure μ, pre-existing mutants are first eliminated from the culture by staining the cells with an antibody specific for CD59 and sorting to collect the upper 50th percentile of the population. This purified GPI(+) population is then returned to culture for 3 weeks, with careful cell counts taken to estimate the number of cell divisions (d) occurring in vitro. After expansion, the frequency (f) of mutants arising in vitro is measured by first incubating with a mixture of mouse antibodies specific for CD48, CD52, CD55, and CD59, followed by a rabbit anti-mouse PE conjugate, followed by a FITC conjugated antibody specific for a transmembrane protein (CD45 or HLA-DR). Live cells are identified based on exclusion of propidium iodide and based on FITC fluorescence; GPI(−) cells are defined as having <4% of the mean PE fluorescence as the GPI(+) population. f is calculated as # GPI(−) cells / total gated live cells. The mutation rate is then calculated as μ = f /d. In initial experiments, 6 separate cultures of P493 were analyzed, demonstrating a median μ of 15 × 10−7 mutations per cell division (range 11 – 33 × 10−7). For comparison, the positive control, a hypermutable mantle cell lymphoma cell line (HBL2), demonstrated a μ of 674 × 10−7, and five BLCLs derived from normal donors demonstrated a median μ value of 37 × 10−7 (range 11 to 57 × 10−7). In parallel experiments, P493 was also grown in the presence of tetracycline and estradiol to reduce MYC: in 5 separate experiments, P493 growing in these conditions demonstrated a median μ of 6 × 10−7. However, in 2 subsequent experiments, we observed a higher μ under “lower MYC” conditions: 81 × 10−7 (SEM ±15 × 10−7) vs 13 × 10−7 (±2.2 × 10−7) and 65 × 10−7 (±6.2 × 10−7) vs 21 × 10−7(±3.3 × 10−7). We then isolated 6 clones of P493: for 5 clones, under “high MYC” conditions, μ was similar to the previous experiments (26 × 10−7, SEM ±3.4 × 10−7), and for 3 of these clones μ went up in the “lower MYC” conditions by 5 to 9 fold. For 2 of these clones, μ was similar in the “high MYC” and “lower MYC” conditions. One of the 6 clones, surprisingly, did not adopt a lower growth rate in the presence of tetracycline and estradiol and demonstrated a reversed pattern: μ was 70 × 10−7 under “high MYC” conditions and 16 × 10−7 under “lower MYC” conditions. We next analyzed 7 cell lines derived from Burkitt's neoplasms which also overexpressed MYC (mean normalized levels ~ 3 × higher compared with normal BLCLs). Here we found a bimodal pattern, as we have previously described for primary acute lymphoblastic leukemia samples: for 4 of the cell lines μ was < 25 × 10−7, whereas 3 samples exhibited μ values comparable to the hypermutable positive control: 229 × 10−7, 562 × 10−7, and 1786 × 10−7 respectively. We conclude that overexpression of MYC is not sufficient to produce hypermutability in human B cells, based on this assay for gene-inactivating mutations. Hypermutability–as detected here–is a common feature of cell lines derived from Burkitt's neoplasms but does not seem to be necessary for its development. Disclosures:No relevant conflicts of interest to declare.