ALBERT

Description: Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

Description: Introduction: Multiple Myeloma (MM) is a genetically complex and evolutionary process with well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. A small number of detectable pre-malignant clones are present in early stage and continue to acquire more genomic abnormalities leading to overt disease. The interaction between cancer cells and their environment is reciprocal, multiple components in the tissue environment can influence cancer clonal evolution and cancer cells in turn can also remodel the microenvironment and further disseminate to spatially separated areas of BM. To accurately predict the course of disease with the presence of BM environment, we require methods to estimate clone-specific growth rates and define clones that have the propensity of dissemination. Methods: We developed a novel 'bone chip' MM metastatic xenograft model using fluorescent protein tagged 'rainbow' system which enables both molecular profiling and functional tracking of clonal dissemination of tumor cells by performing tumor-bearing bone chip implantation subcutaneously to SCID-beige mice (SCID-murine model). Rainbow MM cells with equal proportion of all 15 colors were injected into donor femurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. Tumor clones in the implanted bone chip (primary sites) and distant host BM (metastatic sites) were purified by sorting and underwent RNA sequencing. By intersecting differentially expressed genes, we identified a set of genes, the expression of which were altered during disease dissemination and designated this set of genes as 'metastatic signature'. In addition, we also performed genome-wide CRISPR/Cas9-mediated loss-of-function screen in a subcutaneous xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM. The cell library infected with human sgRNA library was injected subcutaneously into SCID-Beige mice on both flanks. When metastasis was established, the fractions of each sgRNA of the primary and metastatic tumors were calculated to identify genes that facilitate tumor metastasis. Results: We found that the 15 rainbow subpopulations were present with equal distribution in the primary sites but not at the metastatic sites. Confocal imaging showed the difference in cluster structures between primary and metastatic tumors. Most of the clusters in the metastatic sites consisted of cells of single colors. RNA sequencing analysis of two human MM cell lines derived from SCID-murine model demonstrated a distinct gene expression profile of the metastatic tumors. Gene Set Enrichment Analysis of the metastatic signature in publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature is significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Through genome-wide CRISPR screening in vivo, we found that the gene targets of the most enriched sgRNAs in the BM samples were preferentially involved in important cellular processes, such as cell cycle regulation and several oncogenic signaling pathways. Additionally, many sgRNAs that remained the implanted sites until late stage were depleted during dissemination, indicating their targeted genes were important for progression. These depleted sgRNAs mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors. By using a network-based inference of protein activity method, we chose 4 genes (HMGA1, KLF6, TRIM28 and PA2G4) and validated in SCID-murine model using CRISPR mediated loss-of-function screen which prioritized HMGA1 as the key regulator in MM dissemination. Conclusions: Here, we demonstrate that in vivo clonal evolution can be characterized using an in vivo model of MM. The data defines specific subclones that have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. We then established a platform for future invivo CRISPR screens to investigate essential genes of response to targeted therapies and/or immunotherapies. Furthermore, a metastatic gene signature was identified and among these, HMGA1 was validated as potential regulator of MM metastasis. Disclosures Roccaro: AMGEN: Other: Advisory Board; GILEAD: Research Funding. Ghobrial:Takeda: Consultancy; Celgene: Consultancy; BMS: Consultancy; Janssen: Consultancy.

Description: Background This study aimed to determine the benefit of early therapeutic intervention with the combination of elotuzumab, Lenalidomide, and Dexamethasone in patients with high-risk smoldering multiple myeloma (SMM). ClinicalTrials.gov Identifier: NCT02279394. Aims The overarching objective of this trial is to determine progression free survival to symptomatic multiple myeloma (MM). Furthermore, the study examined whether genomic studies can help in determining patients who would benefit the most from this early therapeutic intervention. Methods Patients enrolled in this study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al, Blood 2014. Patients were administered weekly elotuzumab (10 mg/kg) on days 1, 8, 15, and 22 for the first two 28-day cycles while receiving lenalidomide on days 1-21. For cycles 3-8, patients were administered elotuzumab infusions on days 1, 8, and 15. dexamethasone (40mg) was given on days 1, 8 and 15 to 40 of the 50 enrolled patients. After 8 cycles or best response, patients were given the option to mobilize with either cyclophosphamide or plerixafor and collect stem cells for future transplant. Patients were then allowed to continue on maintenance therapy where they were administered elotuzumab (20 mg/kg) on day 1, in combination with lenalidomide days 1-21 of a 28-day cycle. Bone marrow (BM) samples of 32 patients were obtained before starting therapy for baseline assessment and whole exome sequencing (WES) of plasma cells. Results In total, 50 patients were enrolled on this study from January 2015 and completed accrual in December 2016, with the participation of eight sites. The median age of enrolled patients was 62 years (range, 29-79) with 18 males (36%) and 32 females (64%). Interphase fluorescence in situ hybridization (iFISH) detected high-risk cytogenetics (defined by the presence of 17p deletion, t(4;14), and 1q gain) in 20 patients. The median time to response was 2.8 months (range, 1.8-4.6). The most common toxicities were fatigue (92%), followed by diarrhea (72%), and hyperglycemia (62%). The most common grade 3 or more adverse events were hypophosphatemia (34%), neutropenia (26%), and lymphocyte count decreased (22%). Three patients (6%) had grade 4 hypophosphatemia during treatment. Additionally, grade 4 cholecystitis, cataract, lymphocyte count increase, hyperglycemia, neutropenia, and thrombocytopenia occurred in one patient (2%). Diabetic Ketoacidosis and sepsis led to death in a patient (2%). Stem cell collection was successful in all mobilized patients to date. As of this abstract date, the overall response rate is 84% (41/49). There were 3 complete responses (6%), 18 very good partial responses (37%), 20 partial responses (41%), 5 minimal responses (10%), 3 stable disease (6%), and 2 unevaluable patients. All the study participants except for three have finished treatment and are currently under follow up. None of the patients showed progression to overt MM to date. We continue to collect data for progression free survival. WES was performed on 32 samples at the time of initiation of therapy. Recurrent mutations in the MAPK pathway (KRAS, NRAS) and tumor suppressor gene, TP53, were detected in 40% of the cases (16% and 24%, respectively), while mutations in the NF-KB and plasma cell differentiation pathways were present in 13% of patients. Somatic copy number alterations (SCNAs) were called based on WES: 1q duplication, 13q, 17p, and 1p deletions were identified in 25, 31, 12, and 7% of cases, respectively. Interestingly, in 6 patients, high-risk SCNAs (1q gain and 17p deletion) were not reported in iFISH but were detected by WES. The analysis of these 32 samples showed that patients who are harboring mutations in the DNA repair pathway genes, had modest response to treatment. Finally, we are analyzing the transcriptomic profile of CD138 negative cells, which represent the BM microenvironment cells (immune and stromal cells) to characterize the BM microenvironment at baseline and end of treatment, and thus, elucidate the role of these cells in the differential response to therapy. Conclusion The combination of elotuzumab, lenalidomide, and dexamethasone is well tolerated and demonstrates a high response rate with no progression to overt MM to date. Correlation with genomic studies can help define patients who benefit the most from this early therapeutic intervention. Disclosures Ghobrial: Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy. Bustoros:Dava Oncology: Honoraria. Badros:GSK: Research Funding; Celgene: Consultancy, Research Funding; Karyopharm: Research Funding. Matous:Celgene: Consultancy, Honoraria, Speakers Bureau. Rosenblatt:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jakubowiak:Karyopharm: Consultancy, Honoraria; SkylineDx: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding. Zonder:Celgene: Consultancy, Honoraria; Pharmacyclics: Other: DSMC; Janssen: Honoraria; Takeda: Honoraria; Alnylam: Honoraria; Coelum: Honoraria; BMS: Research Funding. Munshi:OncoPep: Other: Board of director. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Celgene: Consultancy. Richardson:Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction: In multiple myeloma (MM), despite well-characterized precursor states such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), there is a lack of sufficient biomarkers to predict mechanisms of disease progression. Most genomic analyses have sought biomarkers by study of the malignant plasma cells, however, cancers form a complex ecosystem with the immune and stromal microenvironment. Thus, to characterize the cellular composition and transcriptional programs of each component of the tumor and microenvironment at different stages of MM progression, we employed a single-cell RNA sequencing on a cohort of 22 patients and 9 healthy donors. Methods: We performed 10X droplet-based single-cell RNA sequencing using CD138-expressing plasma cells and microenvironmental populations isolated from bone marrow (BM) aspirates of patients with MGUS (n=6), low-risk SMM (n=3), high-risk SMM (n=13), newly diagnosed MM (n=8) and from 9 healthy donors (NBM). We collected a total of ~88.8K cells, comprising ~48K CD138+ cells (~36.4 from MM stages) and ~40.8K CD45+/CD138- cells (~30.8 from MM stages).Raw read data was processed using the Cell Ranger pipeline to obtain a gene-by-cell expression matrix, which was used to identify cell types and transcriptional programs by clustering and non-negative matrix factorization. Results: Expression profiles of plasma cells revealed clear tumor-specific differences including known oncogenic drivers in MM (MMSET/FGFR3, CCND1 and MAFB) as well as Lysosome-associated Membrane Protein 5 (LAMP5),Histone Cluster 1 H1 Family Member C (HIST1H1C) and Amphiregulin (AREG) distinguishing them from healthy plasma cells. We identified a subset of cycling plasma cells, observing a range of proliferative activity of the malignant fraction. Furthermore, our approach allowed a unique head-to-head comparison of gene expression changes in normal and malignant plasma cells in the MGUS and SMM patients within an individual, excluding inter-individual variation. We were able to discriminate malignant from non-malignant plasma cells and identify transcriptional alterations including known drivers, genes related to immune modulation (NKBIA) or controlling transcription and differentiation (EID1).Some alterations were patient-specific, while others, such as MHC I overexpression and CD27 loss, were recurrently observed across subsets of the cohort. Analysis of BM microenvironment in several stages of MM progression demonstrated a striking shift in the composition of immune cells with significant infiltration of natural killer cells, non-classical monocytes/macrophages, and T cells, enriched even in the earliest stages of the disease. Further investigation revealed significant upregulation of HLA expression at the mRNA level in CD14+ monocytes/macrophages. Intriguingly, comparison of healthy and patient samples by CyTOF showed downregulation of surface MHC II representation in the corresponding cell type, and moreover, co-culture with MM cell lines induced a sharp decrease of extracellular MHC II. This provided strong evidence for compromised antigen presentation by macrophages in the disease setting, hinting at a mechanism of immune evasion. Additionally, expression signatures in cytotoxic T-cells indicated a substantial skewing towards either granzyme B/H- or granzyme K-expressing memory cell-like transcriptional program. In a subgroup of patients, we found a strong simultaneous enrichment of the anti-viral/anti-bacterial gene expression signature for interferon type-1 activated genes in CD14+ monocytes/macrophages and T cells. Together, our results provide a comprehensive view at the complex interplay of the immune and malignant cells in different stages of the disease. We, for the first time, demonstrate the immune response beginning in premalignant conditions to be heterogeneous, including compromised antigen presentation as well as alterations in cellular composition and signaling. Consideration of the type of immunological response may prove valuable in determination of progression risk, as well as open up potential strategies for therapy. Disclosures Bustoros: Dava Oncology: Honoraria. Ghobrial:Celgene: Consultancy; Janssen: Consultancy; BMS: Consultancy; Takeda: Consultancy.

Description: Introduction. Synergistic delivery of free drugs is highly challenging due to each drug's unique pharmacokinetics and biodistribution profiles. The standard of care Bortezomib, Pomalidomide/Lenalidomide, and Dexamethasone only demonstrates synergy in a specific concentration window. This specific concentration window has been proven difficult to reach using free drugs. Whereas the Bortezomib, Pomalidomide/Lenalidomide, and Dexamethasone drug combination is the standard of care for patients, an improved method to deliver drugs more specifically to the tumor in higher concentration and at their synergistic ratios would greatly improve the clinical outcome of Multiple Myeloma patients treatments. Methods and Results. We developed a novel 10 nm biodegradable bottlebrush polymer made of various PEG macromonomers conjugated to clinically used Myeloma drugs, including Bortezomib, Pomalidomide, Lenalidomide, and Dexamethasone. By combining the different macromonomer-drug conjugates together, we defined the most synergistic drug ratio by using the Chou-Talalay method in MM.1S, and KMS11 cell lines. Before evaluating the efficacy of the combination therapy in-vivo, we determined the maximum tolerated dose (MTD) for the Bortezomib bottlebrush alone by monitoring body weight loss, performing H&E staining in all major organs, and performing blood test panels. We successfully injected up to 25x the tolerated dose of free Bortezomib (0.75 mg/kg once a week) with our novel bottlebrush polymer conjugated to Bortezomib (18.75 mg/kg) without signs of toxicity in healthy Balb/c mice. We next evaluated the efficacy of Bortezomib bottlebrush in an orthotopic model of MM.1SGFP+/Luc+ Multiple Myeloma. We determined by fluorescence imaging of harvested organs and by flow cytometry that approx. 5% of the injected dose of bottlebrush Bortezomib (0.95 mg/kg) accumulated in the bone marrow. As a result, this improved and safer delivery technique allowed us to increase by 12 and 10 days the long-term survival of the MM.1SGFP+/Luc+ SCID mice, as compared to free Bortezomib (0.75 mg/kg) and low dose Bortezomib bottlebrush (0.75 mg/kg), respectively (p-value = 0.038, Mann-Whitney). In order to improve the long-term survival and to reach a complete response, we are currently investigating the use of the combination of the 3 drugs: Bortezomib, Pomalidomide, Dexamethasone simultaneously loaded in our bottlebrush at synergistic ratios and comparing the efficacy and toxicity of this combination to the 3 different single loaded bottlebrushes and to the efficacity and toxicity of the free drug combination. Conclusion. In conclusion, we developed a novel polymer enabling the controlled loading of multiple drugs at their optimal synergistic ratios. In addition, we demonstrated that the conjugation of the drug on our polymer enables the safe delivery of a higher tolerated dose in the body compared to treatments using free drugs and provided improved in-vivo therapeutic outcomes. Disclosures Ghobrial: Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy.

Description: Introduction. Multiple myeloma (MM) is a complex and heterogeneous malignancy of plasma cells that has two precursor states: monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). MGUS and SMM are asymptomatic states that eventually give rise to overt MM, with some patients progressing, while others do not. Recent studies in MM pathobiology have highlighted epigenetic alterations that contribute to the onset, progression and heterogeneity of MM. Global hypomethylation of DNA, including tumor suppressor genes, and hypermethylation of B-cell specific enhancers, abnormal histone methylation patterns due to the overexpression of histone methyltransferases such as MMSET, and deregulation of non-coding RNAs along with mutations in different classes of chromatin modulators underline a potential for epigenetic biomarkers in disease prognosis and treatment. This study aimed to define epigenetic pathways that lead to the dynamic regulation of gene expression in MM pathogenesis. Methods. We performed ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq on 10 MM cell lines and CD138+ plasma cells isolated from bone marrow aspirates of 3 healthy donors, 9 SMM, 8 newly diagnosed MM (NDMM) and 9 relapsed (RRMM) patients. ATAC-seq reads were trimmed of adapters, aligned to hg19 using bowtie2, and filtered for mapping quality 〉=Q30 using the ENCODE ATAC-seq pipeline. Reads mapping to promoter regions, defined as -400 to +250 bases from a refseq transcription start site, were counted using bedtools for each sample. Promoter read counts were then normalized by the total number of reads in promoters in the sample, scaled to 1 million total reads, and converted to log10(x+1) space. Results. To characterize the epigenetic contribution to disease progression in MM, we first identified accessible promoter regions in normal plasma cells (NPC), SMM, NDMM and RRMM patients and found regions displaying differential accessibility in MM progression. Next, we intersected the list of differential accessible regions (DARs) with matched transcriptome data and observed two main clusters: genes with unaltered transcription profiles and genes in which the dynamics of open chromatin regions (OCRs) correlated with gene expression. Transcriptomic analysis revealed that a large portion of the differentially expressed (DE) genes in SMM remain DE in NDMM as compared to NPCs (882 genes out of 1642 and 1150 DE genes in SMM and NDMM, respectively). Those genes were significantly enriched for pathways like epithelial mesenchymal transition, cell cycle checkpoints and mitosis, KRAS signaling and interleukin-JAK-STAT pathways. To investigate the genes that behaved differently among the stages of disease, we looked at differential accessibility and expression in NDMM and SMM samples, and integrated them with Whole-Genome Bisulfite-Sequencing and 450K DNA-methylation data from MM patients and healthy donors (BLUEPRINT). This analysis led to the identification of novel genes in MM progression, such as the transcriptional repressor ZNF254 and IRAK3, a negative regulator of the TLR/IL1R signaling pathway. Although gene expression data for these genes showed comparable mRNA levels in SMM and NPCs, followed by a significant decrease in NDMM/ RRMM, ATAC-seq revealed a striking drop in promoter accessibility in SMM, NDMM and RRMM cases. Comparison of ATAC-seq peaks to DNA methylation and ChIP-seq data revealed that the altered OCR of IRAK3 is actually hypermethylated in MM patients and marked by H3K4me3, a marker of active promoters, in MM cell lines. Hypermethylation of IRAK3 has been described in hepatocellular carcinoma, where it is associated with poor prognosis. Together, our data suggest that the identified IRAK3 OCR may act as a bivalent domain that loses accessibility in the precursor states and gains DNA methylation in MM progression. Hence, IRAK3 methylation could be a novel prognostic marker in MM. Conclusion. We have generated a global epigenetic map of primary tumors from patients at the smoldering, newly diagnosed and relapsed/refractory stage of multiple myeloma. Integrative analysis of ATAC-seq data with DNA methylome, transcriptome and whole-genome map of active and repressive histone marks in our study led to the identification of IRAK3 as a novel epigenetic biomarker of disease progression. Disclosures Licht: Celgene: Research Funding. Ghobrial:Takeda: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

Description: Introduction: Multiple Myeloma (MM) is an incurable malignancy characterized by the proliferation of clonal plasma cells in the bone marrow (BM). MM almost always progresses from the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), which indicates the presence of a gradual clonal evolution underlying progression from the original stages of tumor development to the time of clinical presentation. Clonal heterogeneity adds another layer of complexity to that, by introducing interclonal competition in the context of disease progression or therapeutic bottlenecks. Here we developed a mouse model to investigate the impact of multiple clonal mutations on tumor development, as well as the competitive expansion of individual clones. Methods: Primary mouse MM Vk*Myc cells stably expressing Cas9 were infected with validated sgRNAs to knockout (KO) genes of interest (P53, Cyld, Rb1, Dis3, Prdm1, Traf3 and Fam46c) that are significantly mutated in human MM. KO cells were mixed at a 1:1 ratio with control cells infected with control sgRNA and injected intravenously into 8-week-old RAG2 KO mice. Vk*Myc cells were then isolated from bone marrow and spleen through CD138 positive selection, followed by genomic DNA extraction and NGS sequencing to understand the dynamic changes in abundance of mutants from injection to early and late timepoints. Results: In vitro, most knockout Vk*Myc cells had a similar proliferation rate to control cells with the exception of P53 and Rb1 knockout cells, which grew faster as expected; both P53 and RB1 are known cell cycle regulators. However, when co-injected into RAG2 KO mice (Vk*Myc cells constructed with Cas9 do not engraft in C57BL/6 mice), although P53 and Rb1 knockout cells remained the strongest competitors, occupying the majority of the tumor, most KO cells exhibited significantly enhanced proliferation over control cells. These results indicate that certain mutations only become advantageous in the context of the tumor microenvironment, while mutations that directly affect the tumor cell's proliferation rate give rise to more flexible, potent clones. To better understand these differences, we took advantage of the CRISPR-induced heterogeneous pool of genomic edits per gene, and looked at clonal abundancy rates within each knockout population separately. Interestingly, we found mutants with certain insertions/deletions grew faster than others and were overrepresented at the late stage of disease, even when they were generated from the same double-stranded break. Although it is well established that mutations in different regions of the same gene might have different effects, these results indicate that different mutations in the exact same spot can give rise to clones of variable potency and beg the question of whether mutation sequence is as important as mutation hotspot. Conclusion: We established a mouse model to study clonal competition in vivo, utilizing the CRISPR-Cas9 genome editing toolset. Through our model, we were able to witness a range of competitive potential among genes that are significantly mutated in multiple myeloma, with P53- and RB1-mutants as the strongest competitors. Furthermore, we observed that competitive potential can be conditional, with certain mutants conferring fitness advantage only in the context of tumor microenvironment. Adding another layer of complexity to differential fitness, we found that different mutations in the same spot of the same gene give rise to clones of varied potency, implicating mutation sequence as a novel fitness variable. In this study, we thus demonstrate that mutational candidates can be prioritized based on competitive potential, a process of the utmost importance given multiple myeloma's marked genetic heterogeneity. Disclosures Ghobrial: Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

Description: Clonal evolution drives tumor progression, dissemination and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multi-stage process requires tumor cells to enter the circulation, extravasate and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (Progression through Evolution and Dissemination of Clonal Tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA-sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient datasets. 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR/Cas9 in PrEDiCT model and were shown to be significantly depleted in distant BM sites indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.