Morrow, Zachary

Abstract NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition–based therapies for treating advanced/relapsed MM.

Abstract Background and Aims. Despite the fact that the myeloma mutational landscape was elucidated several years ago, there are yet no approved precision therapies targeting specific driver mutations. The role of common, but somewhat enigmatic, mutational events remains elusive, for example, the loss-of-function mutation of the RNA-processing exosome ribonuclease, DIS3, the third most-common mutation after KRAS and NRAS mutations. Furthermore, complex clonal architectural patterns (such as subclonal BRAF mutations nested within RAS-mutant clones) complicate the design of targeted therapy trials. To generate precision in vivo models that recapitulate the complex clonal and mutational architecture of myeloma, we integrated two powerful technological platforms: the first RAS-driven myeloma in vivo model (VQ model) created through a collaboration between the Zhang and Asimakopoulos labs coupled with flexible avian leukosis virus (ALV)-mediated gene transfer that permits myeloma genome editing in a temporally- and spatially- controlled fashion (Prdm1: TVb-mRFP model)(Asimakopoulos and Varmus, J Virol, 2009; 83(10):4835-43). VQ is based on the state-of-art Vk*MYC model (Chesi et al., Cancer Cell 2008;13(2):167-80). VQ was generated by conditionally activating an NrasQ61R allele in Vk*MYC mice. The resultant VQ model recapitulates an advanced, proliferative and drug-resistant phase of disease characterized by RAS mutations. The transducibility of VQ restored the functionality of the ALV-gene transfer technology. This technology exploits the fact that avian retroviruses cannot transduce mammalian cells unless they are engineered to ectopically express an avian retroviral receptor (in this case, the receptor TVb, driven by a plasma-cell-specific regulatory elements). Recently, the ALV gene transfer technology was adapted to achieve CRISPR-Cas9 mediated gene editing in the tumor niche in vivo (Oldrini et al., Nat Comm 2018;9(1):1466). Approach. We generated TVb-expressing VQ lines (CD138+ B220neg) that home to the bone marrow upon transplantation into Prdm1:TVb-mRFP fully immunocompetent syngeneic recipients in the C57BL6/J background. These VQ cells were engineered to express the nuclease Cas9. To determine the efficiency of gene transfer through ALV vectors (as a prelude to sgRNA delivery), pilot experiments were conducted using two types of ALV vector carrying a fluorescent reporter, GFP (green fluorescent protein): a) an RCAS-type vector that drives reporter expression from the native ALV LTR and b) an RCAN-type vector that drives reporter expression from an internal CMV promoter. Results. ALV titers achieved were routinely in excess of 107-108 infectious units/mL. Parental Vk*MYC cells expressing the avian receptor TVb were poorly transducible with ALV vectors. By contrast, VQ cells expressing TVb were readily transducible. At multiplicity of infection (MOI)=2, 68% of VQ myeloma cells expressed reporter fluorescence after a single round of transduction. At MOI=5 the transduction rate rose to 80%. The mean fluorescence intensity (MFI) positively correlated with MOI, consistent with higher-copy proviral integration at higher MOI. At equivalent MOI, GFP fluorescence from RCAS vectors was more intense than GFP fluorescence driven by an internal CMV promoter in RCAN vectors, suggesting that the ALV LTR is a stronger promoter than CMV in VQ myeloma cells. 100% transduced VQ cells were CD138+B220neg at all conditions tested. Conclusions and Current Focus. We are using ALV-CRISPR technology to generate precision myeloma models to support the next wave of personalized therapy and immunotherapy for this recalcitrant and incurable cancer. Progress on three in vivo models under construction will be presented at the meeting:Subclonal Braf mutation nested within clonal Nras mutation. These models will be treated with next-generation RAF inhibitors (Karoulia Z et al.Nat Rev Cancer 2017;17(11):676-91).Dis3 loss-of function mutation in constellation with co-clonal Ras mutation. The pathogenic consequences of Dis3 disruption remain poorly understood as no parallels exist in other cancers.The murine homolog of the t(4;14) translocation. We are targeting breakpoint cluster regions within perfectly syntenic sequences on mouse chr. 5 (containing Fgfr3 and Mmset loci) and mouse chr.12 (containing the murine Igh locus). Disclosures No relevant conflicts of interest to declare.