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Rapid advances in technology are revealing previously unknown organization, cooperation, and limitations within the population of nontumor cells surrounding the tumor epithelium known as the tumor microenvironment (TME). Nowhere are these findings more pertinent than in the gastrointestinal (GI) tract where exquisite cell specialization supports a complex microenvironmental niche characterized by rapid stemness-associated cell turnover, pathogen sensing, epithelial orchestration of immune signaling, and other facets that maintain the complex balance between homeostasis, inflammation, and disease. Here, we summarize and discuss select emerging concepts in the precancerous microenvironment, TME, and tumor epithelial-TME crosstalk as well as their implications for the management of GI cancers.

With the launch of the National Cancer Institute’s Precision Medicine Initiative in 2015, there has been a shift to trial designs that tailor health care solutions to individual patients by using a screening platform and by moving away from the one-trial/one-biomarker-at-a-time approach. To make precision medicine a reality, it is critical to identify and validate potential biomarkers to help select patients who will truly benefit from a targeted therapy. In this article, we discuss five trial designs: enrichment, umbrella, basket, subgroup, and window of opportunity. For each trial design, we describe the design characteristics, use ongoing or completed trials as case studies, provide any recent advances to the trial design, and discuss advantages and disadvantages of each design.

Abstract Objective To design, develop, and evaluate a scalable clinical data normalization pipeline for standardizing unstructured electronic health record (EHR) data leveraging the HL7 Fast Healthcare Interoperability Resources (FHIR) specification. Methods We established an FHIR-based clinical data normalization pipeline known as NLP2FHIR that mainly comprises: (1) a module for a core natural language processing (NLP) engine with an FHIR-based type system; (2) a module for integrating structured data; and (3) a module for content normalization. We evaluated the FHIR modeling capability focusing on core clinical resources such as Condition, Procedure, MedicationStatement (including Medication), and FamilyMemberHistory using Mayo Clinic’s unstructured EHR data. We constructed a gold standard reusing annotation corpora from previous NLP projects. Results A total of 30 mapping rules, 62 normalization rules, and 11 NLP-specific FHIR extensions were created and implemented in the NLP2FHIR pipeline. The elements that need to integrate structured data from each clinical resource were identified. The performance of unstructured data modeling achieved F scores ranging from 0.69 to 0.99 for various FHIR element representations (0.69–0.99 for Condition; 0.75–0.84 for Procedure; 0.71–0.99 for MedicationStatement; and 0.75–0.95 for FamilyMemberHistory). Conclusion We demonstrated that the NLP2FHIR pipeline is feasible for modeling unstructured EHR data and integrating structured elements into the model. The outcomes of this work provide standards-based tools of clinical data normalization that is indispensable for enabling portable EHR-driven phenotyping and large-scale data analytics, as well as useful insights for future developments of the FHIR specifications with regard to handling unstructured clinical data.

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.

Abstract Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Abstract Introduction REGN1979 is an anti-CD20 x anti-CD3 bispecific IgG4 antibody (Ab) modified to reduce Fc binding. Engaging both targets results in CD20-specific, local T-cell activation and cytotoxicity, a mechanism of action distinct from standard anti-CD20 Abs. We report updated promising efficacy results of a Phase 1 trial of REGN1979 in patients (pts) with relapsed/refactory (R/R) B-NHL previously treated with anti-CD20 Abs. Methods The primary objectives of the study are to determine safety, tolerability, and occurrence of dose limiting toxicities (DLTs). Other objectives include assessment of preliminary antitumor activity, pharmacokinetics (PK), and pharmacodynamics. Eligible pts with R/R NHL must have received at least 1 prior CD20-directed therapy. Treatment consists of 12 weekly doses of REGN1979 followed by every 2 week dosing for 12 doses for a total of 36 weeks. Guidelines are provided for management of cytokine release syndrome (CRS) and include steroids and/or tocilizumab at investigator discretion. Results As of June 1, 2018, 54 pts with B-NHL were treated with REGN1979 monotherapy: DLBCL (pt number [n]=30), FL (n=16), MCL (n=5), MZL (n=2), and WM (n=1). The median number of prior regimens was 3 (range, 1-11); 41 pts were refractory to their last prior systemic therapy, 18 had bulky disease, and 6 had prior HSCT. Pts were treated with REGN1979 0.03-27 mg and received a median of 7 (range, 1-24) doses. Eight pts remain on treatment, 13 completed treatment, and 33 discontinued therapy prior to the planned 36 wks (majority [n=22] due to progressive disease [PD]). There have been no DLTs to date. The most common treatment-related treatment-emergent adverse events (TR-TEAEs) included infusion-related reactions (IRR) or CRS; 26 pts experienced CRS (Grade 1-2, n=23; Grade 3, n=3) with a median duration of CRS of 2 (range 1-15) days. Six pts received tocilizumab. The severity of CRS symptoms declined through optimized pre-medication even with REGN1979 dose escalation. Other common Grade ≥3 TR-TEAEs were lymphocytopenia/ decreased lymphocyte count (n=8); neutropenia/ decreased neutrophil count (n=7); and thrombocytopenia/ decreased platelet count, hypotension, hypophosphatemia, and anemia (each n=3). Seventeen pts experienced a nervous system event including headache, dizziness, paraesthesia, dysgeusia, and peripheral neuropathy with no Grade ≥3 events; no neurologic event required termination of study drug. Seven pts died on study: PD (n=5), gastric perforation (n=1), and cardiac arrest (n=1). TEAEs leading to premature discontinuation of REGN1979 were Grade 3 fatigue (n=1), Grade 3 hemolysis (n=1), and Grade 2 pyrexia/Grade 2 tachycardia (n=1). Among 27 pts treated with REGN1979 ≥5 mg (dose associated with tumor killing in pre-clinical data), the overall response rate (ORR) was 55.6% (5 complete response [CR] and 10 partial response [PR]; Table/Figure). Responses were seen in 7/7 FL Grade 1-3a pts (5 CR, 2 PR), 6/15 DLBCL pts (all PR), 2/2 MCL pts (all PR). At 18 mg and 27 mg of REGN1979, 4 of 4 pts with DLBCL had best response of PR. Post data cut-off, 1 pt with a DLBCL best response of PR converted to CR. PK and pharmacodynamic assessments suggest that for pts treated with REGN1979 5-18 mg, best exposures in the first 3 weeks appear to linearly increase with dose. In pts treated with up to REGN1979 18 mg, a maximum mean Ctrough of 1.6 mcg/mL was observed in the first 3 weeks of weekly dosing. Studies of peripheral blood biomarkers demonstrated depletion of peripheral B lymphocytes, transient margination of circulating T-cells, and elevated circulating cytokines following REGN1979 dosing. Increased peak cytokine levels (IL-6, IL-10, and TNF-alpha) were observed in pts with CRS. Immunohistological analysis of malignant lymph node tissue demonstrated a decrease of CD20 expression in responding pts; among the responders, subsequent relapse was associated with either maintenance of CD20 expression or further CD20 loss, suggesting antigen-dependent and independent disease escape mechanisms. Conclusions REGN1979 displays efficacy and an acceptable safety profile in pts with R/R B-NHL. Most TR-TEAEs were IRR/CRS and have been well managed with supportive care. No significant neurological toxicity has been observed. At doses of 5-27 mg of REGN1979, the preliminary ORR was 100% in pts with FL Grade 1-3a and 40.0% in pts with DLBCL. This promising efficacy warrants further clinical investigation. Disclosures Bannerji: Regeneron Pharmaceuticals, Inc.: Consultancy; AbbVie, Inc.: Consultancy. Arnason:Regeneron Pharmaceuticals, Inc.: Consultancy. Advani:Bayer Healthcare Pharmaceuticals: Other: Consultancy/Advisory Role; Janssen Pharmaceutical: Other: Institutional Research Support; Bristol Myers Squibb: Other: Consultancy/Advisory role and Institutional Research Support; Pharmacyclics: Other: Institutional Research Support; Takeda: Other: Consultancy/Advisory Role; Millenium: Other: Institutional Research Support; Gilead/Kite: Other: Consultancy/Advisory Role; Infinity: Other: Institutional Research Support; Merck: Other: Institutional Research Support; Forty Seven, Inc: Other: Institutional Research Support; Cell Medica: Other: Consultancy/Advisory Role; Agensys: Other: Institutional Research Support; Celgene: Other: Institutional Research Support; Seattle Genetics: Other: Consultancy/Advisory role, Institutional Research Support; Kura: Other: Institutional Research Support; Roche/Genentech: Other: Consultancy/Advisory Role, Institutional Research Support; Kyowa: Other: Consulting/Advisory Role; Regeneron Pharmaceuticals, Inc.: Other: Institutional Research Support; Autolus: Other: Consultancy/Advisory Role; AstraZeneca: Other: Consultancy/Advisory Role. Brown:Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Verastem: Consultancy, Research Funding; Pharmacyclics: Consultancy; Celgene: Consultancy; Genentech: Consultancy; TG Therapeutics: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Abbvie: Consultancy; Gilead: Consultancy, Research Funding; Sun Pharmaceutical Industries: Research Funding; Sunesis: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Boehringer: Consultancy. Allan:Genentech: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy. Ansell:Bristol-Myers Squibb: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Merck & Co: Research Funding; LAM Therapeutics: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Celldex: Research Funding; Takeda: Research Funding; Pfizer: Research Funding. O'Brien:Kite Pharma: Research Funding; Aptose Biosciences Inc.: Consultancy; Pharmacyclics: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Astellas: Consultancy; Sunesis: Consultancy, Research Funding; Alexion: Consultancy; Amgen: Consultancy; TG Therapeutics: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Vaniam Group LLC: Consultancy; Regeneron: Research Funding; Janssen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Acerta: Research Funding; Gilead: Consultancy, Research Funding. Chavez:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Research Funding; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Humanigen: Consultancy. Lowy:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Charnas:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Sternberg:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Ambati:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Adriaens:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Ufkin:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Yan:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Li:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Navarro:Regeneron Pharmaceuticals, Inc.: Employment. Gasparini:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Jankovic:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Fiaschi:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Zhang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Hamon:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Thurston:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Topp:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Research Funding; Regeneron Pharmaceuticals, Inc.: Honoraria, Research Funding.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients. To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients. We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 64 Megakaryocytes are one of the few cell types that undergo a modified form of the cell cycle termed endomitosis, in which cells skip the late stages of mitosis to become polyploid. Murine and human megakaryocytes commonly reach modal ploidy states of 32N and 16N, respectively, and can sometimes achieve DNA contents as high as 64N. Polyploidization is associated with upregulation of megakaryocyte lineage specific genes, proplatelet formation and expression of genes related to apoptosis. RNA expression array studies have shown that high ploidy states are strongly correlated with megakaryocyte differentiation and maturation. Importantly, the choice of a megakaryocyte to undergo polyploidization and differentiation is inextricably linked to exit from the proliferative cell cycle. Given that megakaryocytes in patients with essential thrombocythemia are hyperproliferative and that those in primary myelofibrosis fail to undergo normal differentiation or polyploidization, we hypothesized that small molecule inducers of polyploidization would drive these cells to exit the proliferative cell cycle and undergo terminal differentiation or death. In collaboration with the Broad Institute, we performed a high throughput screen and identified small molecules that induce polyploidization and proliferative arrest of malignant megakaryocytes, including those that express MPLW515L and JAK2 V617F. We have shown that these compounds, including the Rho kinase inhibitor dimethylfasudil (diMF), selectively increase polyploidization, expression of megakaryocyte cell surface markers, and apoptosis of murine and human megakaryocytic cell lines and primary cells. Furthermore, diMF blocked the growth of primary human AMKL blasts both in vitro and in vivo. With respect to MPNs, diMF showed potent activity against megakaryocytic cell lines and primary cells expressing either JAK2 V617F or MPL W515L alleles commonly associated with these disorders. diMF inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis of megakaryocytes transduced with JAK2 V617F or MPLW515L. diMF also significantly reduced megakaryocyte colony forming units (CFU-MK), and induced polyploidization and differentiation of bone marrow and fetal liver megakaryocytes from Gata1 mutant mice, which develop a PMF-like disease. Given that diMF induces growth arrest, polyploidization and apoptosis of cells that express activated mutants of JAK2 and MPL, we predicted that it, as well as other small molecule inducers of polyploidy, would be efficient at restraining aberrant megakaryocyte proliferation in both PMF and ET. To assay the effectiveness of diMF in these disorders, we treated peripheral blood mononuclear cells from patients with either PMF or ET with diMF and monitored growth and maturation of megakaryocytes. We discovered that diMF induced polyploidization and subsequent apoptosis of both types of MPN primary samples. diMF also reduced CFU-MK of these MPN patient samples. Next, we assessed the activity of diMF in a model of MPN in which congenic recipients of MPLW515L transduced Balb/C bone marrow cells develop a rapid MPN characterized by leukocytosis, thrombocytosis, bone marrow fibrosis, and death. diMF led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen of a mouse model of PMF. diMF also led to a significant reduction in the platelet count and a trend towards decreased white cell count, with no effect on hematocrit. Overall, diMF results were comparable to intermediate doses of INCB16562. These encouraging results strongly suggest that diMF induces a decline in megakaryocyte lineage, which leads to reduction in platelet count, and support pre-clinical development of diMF for megakaryocytic subtypes of MPNs. Of note, diMF did not inhibit the phosphorylation of Stat5 or Stat3, suggesting that it acts through a mechanism distinct from JAK2 inhibitors. Interestingly, combination of diMF with a selective JAK2 inhibitor greatly enhanced the efficacy of diMF to inhibit proliferation and induce apoptosis in MPLW515L transduced megakaryocytic cell line. These data support combining JAK inhibition and induction of megakaryocyte polyploidy as a new therapeutic strategy for MPNs. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3862 Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs). Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion. Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.