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MiRNAs are small noncoding RNAs that control gene expression by binding to cognate sites in the 3′ untranslated region (3′ UTR) of target messenger RNAs (mRNAs). MiRNAs are transcribed as primary (pri) mRNAs and processed into precursor (pre) miRNAs by nuclear DROSHA before being exported into the cytoplasm via the Exportin 5 (XPO5)/RAN-GTP complex. Once in the cytoplasm, pre-miRNAs are further processed by Dicer into mature miRNAs. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML) and confers growth and survival advantages to leukemia blasts. Among miRNAs, miR-155 has emerged as one of the most significantly upregulated in the FLT3-ITD+ AML and has been shown to play a pivotal role in uncontrolled blast hyperproliferation and survival. Herein, we report a previously unrecognized activity of FLT3-ITD that leads to deregulation of miR-155 expression by providing the evidence for the existence of FLT3-dependent non-canonical mechanisms of miRNA biogenesis. We initially observed that FLT3-ITD blocked the biogenesis of intronic miRNAs via inhibition of the "gatekeeper" XPO5/RAN-GTP complex that allow nucleus-to-cytoplasm pre-miRNA transport. By using in vitro phosphorylation assay with [32P]-ATP labeling, we demonstrated that FLT3-ITD phosphorylates a Sprouty related EVH1 domain-containing protein 1 (SPRED1), and that phospho-SPRED1 in turn inhibited the XPO5/RAN-GTP complex thereby halting transportation of pre-miRNAs from the nucleus to cytoplasm. This resulted in a decrease of several intronic miRNAs involved in normal hematopoiesis (i.e., miR-29b, miR-181a, miR-146b, miR-126). Accordingly, knocking down of SPRED1 expression in FLT3-ITD+ AML cells resulted in increased production of mature intronic miRNAs. Since the XPO5/RAN-GTP complex is the main gatekeeper for miRNA biogenesis, these results appeared in contradiction with the miR-155 upregulation which has been invariably observed in FLT3-ITD+ AML blasts. Differently from intronic miRNAs, miR-155 is hosted at the genomic site of long non-coding RNA (lnc-RNA) and we therefore hypothesized that it follows a different path of biogenesis. In fact forced expression of FLT3-ITD in lin-Sca1+kit+ (LSK) cells decreased intronic miRNA biogenesis (i.e., miR-126) and increase production of lnc-RNA hosted miRNAs (i.e., miR-155). Accordingly, we demonstrated that upon FLT3-ITD activation, AKT phosphorylated DDX3X, a DEAD-box RNA helicase protein thereby reducing its ability to bind heterogeneous nuclear Ribonucleoprotein U (hnRNP U), a component of the hnRNP complex associated with pre-mRNA splicing. Consequently, in FLT3-ITD cells, BIC-155 splicing is decreased. In fact, we measured significant increased levels of phospho-DDX3X and unspliced BIC-155 in primary FLT3-ITD+ blasts compared with FLT3-ITD- AML blasts. In consistent, DDX3X knock-down (KO) in FLT3-ITD- cells decreased BIC-155 splicing whereas DDX3X re-expression reversed these effects. The excess of unspliced BIC-155 RNA then bound to nuclear RNA export factor 1 (NXF1), a shuttle protein that transports poly A+ RNAs from the nucleus to the cytoplasm. In consistent, NXF1 KO blocked unspliced BIC-155 nucleus-to-cytoplasm transportation. In FLT3-ITD+ blasts, once in the cytoplasm, the unspliced BIC-155 RNA was then processed by cytoplasmic isoforms of DROSHA, as demonstrated using immunostaining, RNA Immunoprecipitation (RIP), and other gain- and loss-of-function experiments. Furthermore, overexpression of cytoplasmic DROSHA but not nuclear DROSHA isoform increased the processing of unspliced BIC-155 to mature miR-155. None of the intronic miRNAs studies interacted with cytoplasmic DROSHA. The bound of BIC-155 RNA with cytoplasmic DROSHA was unique of FLT3-ITD+ blasts, as it was not observed in FLT3-ITD- blasts. Thus, our results indicate a two-fold activity of FLT3-ITD that leads to decreased levels of intronic miRNAs via XPO5/RAN-GTP blockage and upregulation of other lnc-RNA hosted miRNAs via cytoplasmic DROSHA activation. The net result is suppression of intronic miRNAs that participates to the regulation of normal hematopoiesis and upregulation of lnc-RNA-hosted miRNA, especially miR-155 that contribute to aberrant blast hyperproliferation in the FLT3-ITD AML phenotype (Figure 1). Disclosures No relevant conflicts of interest to declare.

Introduction: Daratumumab (Dara) is a human monoclonal antibody targeting the highly expressed multiple myeloma (MM) surface receptor CD38, with significant activity in relapsed MM. However, resistance to Dara develops in virtually all patients (pts). Thus, in order to maximize its clinical activity and prevent resistance, it is imperative to understand why pts stop responding. Our group recently reported that, in addition to the malignant cells, several immune-effector cells also express CD38. Thus, we hypothesized that the expression of CD38 or lack of it on non-cancer immune cell-subsets may also play a role in both clinical response and resistance to Dara. Results: To address our hypothesis, we analyzed CD38 surface expression in CD138+ MM cells isolated from 5 Dara-naïve (D-naïve) pts and from 8 Dara progressing pts (D-prg) who had discontinued Dara therapy for at least 4 weeks prior to analysis. We observed that the CD138+ MM-cells of D-prg displayed a lower median fluorescence intensity (MFI) of CD38 compared to the MFI of MM cells isolated from the D-naïve (p=0.04); no significant difference was however observed in the percentage of CD38+ MM cells between the two groups. In fact, all of the MM cells expressed CD38 in both D-naïve and D-prg, supporting the hypothesis that resistance to Dara was not mediated by loss of CD38 expression on MM cells. Next, we looked at whether CD38 on the surface of MM cells from D-prg was still recognizable by Dara. Thus, we treated MM cells ex-vivo with Dara at the clinically achievable concentration of 100 μg/ml. We showed that Dara was still able to bind CD38 on the surface of the D-prg CD138+ MM cells, since, after Dara treatment, no CD38+ signal was detected upon staining with a CD38 monoclonal antibody sharing the same targeting CD38 epitope (clone IB6) (Fig.1a). Conversely, we detected a CD38+ signal when a different anti-human CD38 multi-epitope antibody was used in a similar experiment (Cytognos) (Fig.1b). CD38 mRNA expression was observed in all MM cells isolated from D-prg, and we were not able to identify any missense mutations in CD38 mRNA from MM cells of D-prg for whom material was available for analysis. Thus, having demonstrated that resistance to Dara was not related to the expression of CD38 on the MM-cells, we turned our attention to the MM bone marrow (BM) microenvironment (ME). We performed RNA-sequencing of the BM-ME depleted of the CD138+ MM cells obtained from the D-prg (n=5) and D-naïve (n=5). A gene expression signature differentiated the two groups. In D-prg, we observed a significant downregulation (fold-change Conclusions: Our data are supportive of the hypothesis that loss of Dara binding to MM-cell surface CD38 or the complete loss of CD38 on MM cells may not necessarily be the cause of Dara treatment resistance. We instead propose that Dara binding on CD38+ immune effector cells may be critical and that the loss of CD38 expression on these cells can lead to mechanisms of Dara-acquired resistance. Disclosures Rosenzweig: Celgene: Speakers Bureau. Krishnan:Celgene: Consultancy, Equity Ownership, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Sutro: Speakers Bureau; Takeda: Speakers Bureau; Onyx: Speakers Bureau.

Inv(16)(p13q22)/t(16;16)(p13.1;q22) is one of the most common chromosomal mutations found in human acute myeloid leukemia (AML). This inversion on chromosome 16 creates a fusion gene CBFb-MYH11 and leads to the expression of a fusion protein CBFβ-SMMHC. Expression of Cbfβ-SMMHC (CM) in a conditional Cbfb-MYH11 knock-in mouse model (Cbfb+/56M/Mx1-Cre) leads to development of spontaneous AML with the acquisition of additional genetic and epigenetic alterations. In order to further understand the underlying mechanism(s) driving leukemogenesis and identify predictive biomarkers during disease progression, we performed RNA-seq to track changes of transcriptiome in time series peripheral blood samples using illumina HiSeq sequencer. A cohort of 16 mice were included in the experiment, 9 out of which are CM conditional knock-in mice and the remaining 7 are controls. Peripheral blood samples were collected before the induction of CM expression as the "0" time point samples. After induction, peripheral blood samples were collected at 1, 2, 3.5, 4.5, 5.5 and 6.5 month (experimental end point) after induction, and leukemic samples were collected when mice were moribund as the "end" time point samples. Between 3.5 and 6.5 months, 6 out of 9 CM knock-in mice in this cohort had succumbed to lethal leukemia. Total RNA was isolated for mRNA and microRNA (miR) sequencing library preparation. The mapped sequencing read counts were annotated to genes and differential expression was compared using edgeR. False discovery rate (FDR) were used to adjust for multiple comparisons. Genes with adjusted P value (FDR) 2 were considered differentially expressed genes. Unsupervised clustering analysis revealed that all diseased mice showed unique disease-related mRNA and miRNA signatures at the end point. The disease-related signatures were absent in all control mice and the three CM conditional knock-in mice that did not develop leukemia. We found that 2,032 mRNA genes and 106 miRs were significantly up-regulated whereas 2,926 mRNA genes and 121 miRs were significantly down-regulated at the leukemia end point. Principal component analysis revealed that the disease-related changes occur quite early, at 1-2 month after induction. Among the earliest changes, miR-126-3p and miR-126-5p, previously reported to be associated with human inv(16) AML, were significantly up-regulated (log fold change =2.09 and 2.36 respectively; p< 0.0001) at 1 month and increased progressively throughout leukemia progression (log fold change = 4.11 and 4.45; p< 0.0001). DAVID gene ontology (GO) analysis of genes negatively correlated with miR-126 expression showed significant enrichment for mitotic cell cycle and cell division GO pathways. In addition, dynamic changes in a number of predicted miR-126 targets were seen during disease progression. We reasoned that high miR-126 expression might contribute to AML progression. Lentiviral miR-126 knock-down in AML LSK (Lin-/Sca1+/cKit+) cells from CM induced mice led to increased apoptosis and reduced quiescence in vitro. To assess the role of miR-126 in leukemia progression in vivo, we then designed a novel CpG-miR-126 inhibitor (Zhang et al, submitted). Mice transplanted with CM+ AML and treated with with anti-miR126 (5 mg/kg, daily, 30 days) had a significant delay in leukemia progression and enhanced survival (control 42% survival v.s. anti-miR-126 80% survival at 70 days; n=7-10), supporting the notion that high miR-126 contributes to disease growth. Further mechanistic investigation of miR-126 function during inv(16) AML progression are ongoing. In conclusion, transcriptome analysis of time series samples during spontaneous leukemia progression allows identification of disease-related changes and early increase of miR-126 as a potential biomarker. Further functional validation is expected to provide mechanistic insights and rational for targeting miR-126 in inv(16) AML. Disclosures Stein: Seattle Genetics: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Stemline Therapeutics: Consultancy, Research Funding; Argios: Research Funding; Celgene: Research Funding.