Gyotoku, Kelli

Abstract Multiple myeloma (MM) is the second-most common hematological malignancy in the United States. Myeloma tumors have significant genetic heterogeneity that impacts disease progression and promotes resistance to treatment. RAS is a key oncogenic driver that activates the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)/AKT pathways..Mutations in genes that code for RAS isozymes, KRAS, HRAS, or NRAS, lead to increased levels of activated GTP-RAS and are associated with the most aggressive forms of cancer. Mutations in the RAS family of genes are most common (40-50%) in myeloma. We and others have reported that RAS mutations may not be disease-initiating but rather arise during disease progression and relapse or as subclonal events that impart selective growth advantage and emerging drug resistance. In addition, non-mutant RAS can also be hyperactivated from upstream signaling (like receptor tyrosine kinases) or loss-of-function mutations in GTPase-activating proteins (GAPs). Given the importance of RAS mutations, allele-specific RAS inhibitors have long been considered an attractive strategy for myeloma treatment. However, multiple studies have reported various mechanisms of resistance to allele-specific RAS inhibitors as well as isoform-specific pan-RAS inhibitors, including newly acquired activating RAS mutations, RAS amplification, unchecked activation of co-expressed wild-type RAS (WT-RAS) isozymes, or acquired bypass mechanisms like new oncogenic fusions. Since MM remains incurable, we sought to improve upon currently available treatments by developing small-molecule inhibitors that preferentially inhibit myeloma cells with activated RAS isozymes. We have created NRasQ61X and KRasG12X mutations by CRISPR-Cas directed edits in a single endogenous RAS allele in human myeloma cell lines (HMCLs) that initially had two wild-type alleles. We found that these mutant-NRAS and mutant-KRAS edited cells have increased proliferation compared to cells with WT-RAS, as well as acquired resistance to several commonly used therapeutics. In this study, we used a triangulation method that combines our drug combination candidate prediction algorithm called ‘secDrug’ and the ‘Direct to Drug’ screening platform that evaluated 84 FDA-approved oncology drugs and emerging therapeutics in these RAS mutant myeloma lines. We identified BIRC5 as a potential target for RAS-mutant myeloma. BIRC5 expression is a key mechanism for the evasion of apoptosis. Notably, oncogenic RAS has been shown to promote MYC stabilization via upregulation of BIRC5. Earlier studies suggested that BIRC5 inhibitors are potent in targeting drug-resistant subclones in hematological malignancies. We performed in vitro cytotoxicity studies and cell-based assays using an in-house panel of HMCLs (n>50) representing WT- and mutant-RAS, as well as innate and acquired response/resistance to the standard of care drugs, proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), to confirm the potency of novel selective BIRC5 inhibitors, as single agent (IC50 range 0.22-15.30 nM; Mean 4.431±1.041 nM) and in combination with PIs and IMiDs (Chou-Talalay's Combination Index or CI <0.3, indicating strong synergism). Further, we used bulk and single-cell whole-transcriptomics, siRNA-mediated gene knockdown studies, and functional validation to characterize the treatment-induced genes and molecular pathways underpinning the mechanism of action and drug synergy at the subclone level. Finally, we compared the efficacy of these BIRC5 inhibitors against several RAS inhibitors, including cyclorasin B4-27, a bicyclic peptidyl pan-RAS inhibitor, RMC-6236, a multi-selective noncovalent RAS(on) inhibitor, and ADT-007, a novel, highly potent, and selective pan-RAS inhibitor that we have synthesized, and observed comparable effects on cell death and apoptosis. Ex vivo analysis in bone marrow-derived CD138+ cells from myeloma patients using high-throughput Mass Cytometry (CyTOF or Cytometry by Time-Of-Flight) confirmed the dose-dependent reduction of MCL1, Ki-67, CCND1, c-KIT, and MYC. We will present these results that demonstrate the clinical potential of BIRC5 inhibitors as novel candidates in curbing progression and drug resistance in RAS-mutant myeloma.

Myeloma is well known to have significant genetic heterogeneity among patients' tumors, affecting both progression as well as therapeutic responses. Moreover, subclonal diversity can lead to selective outgrowths that are influenced by new mutations, as well as the bone marrow microenvironment and therapeutic exposures. The ras family of genes (N -and K-) are the most common mutations (40-50% ) in myeloma. Notably, ras mutations occur among a variety of other co-existing chromosomal abnormalities. Ras mutations were found to be more commonly associated with cyclin D1 high expression (e.g., resulting from 11;14 translocations). We and others have also shown that rasmutations may not be disease-initiating but may arise during disease progression and relapse; or are subclonal events that impart selective growth advantage and emerging drug resistance. There is evidence that activating mutations (codons 12,13, 61) in K- ras may have different clinical impact than activating mutations in N- ras yet there are conflicting clinical outcomes that may be due to therapies analyzed or differences in other co-existing genetic mutations. Previous studies using transfection of genes with targeted mutations show random integration and often in multiple copies, thus, limiting interpretations. We chose to create N ras61 and K ras12 mutations by CRISPR-cas directed edits in a single endogenous ras allele in a myeloma cell line (FLAM76) that initially had two wild type alleles (and an 11;14 translocation). We then compared the impact of these edits on growth, signaling, gene expression, and drug responses. We find both the mutN ras and mutK ras edited cells have increased proliferation compared to wild type. However, we find that the N ras mutation increases MAPK signaling and downstream gene activation to a greater extent than the K rasmutation. Based on RNAseq analysis, a MAPK gene activation score was higher in mutN ras edited cells than mutK ras edited cells. Further, we see distinct gene expression patterns between mutN ras versus mutK ras edited cells (See heat map Fig) impacting distinct pathways. Notably, mutNras cells have much higher NOXA expression, increasing sensitivity to apoptosis-inducing drugs (e.g., proteasome inhibitors); and Kras induces anti-apoptotic Bcl-xL, reducing sensitivity. We have conducted a high throughput drug screen with 84 drugs, and while some drugs are active against both mutN ras and mutK ras edits (Proteasome inhibitors), we have identified drugs with differential response to K versus N ras mutations. For example, mutK ras FLAM76 cells are more resistant to proteasome inhibitors; and particularly more resistant to HSP inhibitors and HDAC inhibitors compared to wild type or mutN ras cells. In contrast, mutK ras cells are more sensitive to PikFyve and IL-12 inhibitors than mutNras or wild type cells. Gene expression analysis shows that mutKras cells result in more downregulation of apoptotic genes and higher upregulation of cell survival genes, as well as ER stress pathway genes. Our results suggest that treating myelomas containing K versus N ras activating mutations may benefit from understanding distinct pathway activations and targeting selective drug therapies. We are currently testing new pan-Ras inhibitors to determine if there are distinct responses between our N- and K- ras edited cells, and we are expanding testing to a wider panel of myeloma cell lines containing ras mutations in the context of other genetic abnormalities. Details of gene expression distinctions, pathways, and drug responses that distinguish mutN ras and mutK ras in myeloma will be presented.