Yoshimi, Akihide

Abstract RNA-binding proteins (RBPs) regulate many aspects of transcription and translation in a cell- and tissue-specific manner and are frequently dysregulated in malignancy. We systematically evaluated RBPs preferentially required in acute myeloid leukemia (AML) over other forms of cancer or normal hematopoietic precursors using a CRISPR/Cas9 domain-based, loss-of-function screen targeting 490 classical RBPs with 2,900 sgRNAs (Fig. A). This screen was performed in cells lines representing AML, T-cell acute lymphoblastic leukemia (T-ALL), and lung adenocarcinoma (LUAD) and revealed multiple RBPs preferentially required for AML survival, but not for T-ALL or LUAD survival. We identified genes encoding 21 RBPs that were >3-fold depleted in AML cells and significantly overexpressed in AML patient samples versus normal adult CD34+ precursors (p-value < 0.05; Fig. B). Amongst RBPs required and upregulated in AML was RBM39, an RBP described to be involved in a number of cellular processes and to interact with key splicing proteins SF3B1 and U2AF2. Genetic ablation of Rbm39 in mouse MLL-AF9 leukemia cells dramatically delayed AML development and progression (Fig. C). In parallel, it has recently been described that a class of clinically-validated anti-cancer sulfonamide compounds (including indisulam and E7820) mediate RBM39 degradation as their dominant cellular mechanism of action. This occurs via novel interactions with the DCAF15 adapter protein of the CUL4/Ddb1 ubiquitin ligase complex with RBM39 as a neo-substrate. Treatment of MOLM-13 cells xenografted into mice with indisulam conferred significant anti-leukemic effects and improved overall survival (Fig. D). To explore the mechanism of RBM39 dependence in AML, we performed proteomic analyses of RBM39 interacting proteins in MOLM-13 cells as well as transcriptome-wide analysis of RBM39 RNA binding by enhanced UV cross-linking and immunoprecipitation (eCLIP) in the same cells. RBM39 physically interacted with an entire network of RBPs identified by our CRISPR screen as crucial for AML cell survival in addition to interacting with the core SF3b splicing complex. Further, anti-RBM39 eCLIP revealed RBM39 binding to exonic regions and most enriched at exon/intron borders at 5' and 3' splice sites of pre-mRNA (Fig. E), suggesting a prominent role of RBM39 in regulating splicing. Consistent with this, RNA-sequencing of AML cells following RBM39 deletion revealed significant effects of RBM39 loss on RNA splicing, most prominently causing increased cassette exon skipping (Fig. F). Recent studies suggest that myeloid leukemias with mutations in RNA splicing factors are sensitized to pharmacologic perturbation of RNA splicing. Analysis of the effects of RBM39 degrading compounds over a panel of 18 AML cells revealed that leukemia cells bearing splicing factor mutations or with high DCAF15 expression were the most sensitive to treatment (Fig. G). Genetic introduction of SF3B1, SRSF2, or U2AF1 hotspot mutations in K562 or NALM6 cells resulted in a 20-50% reduction in IC50 in response to sulfonamides. We next performed RNA sequencing of isogenic K562 cells with or without knockin of SF3B1K700E and SRSF2P95H mutations into the endogenous loci, and treated at the IC50 of E7820 or E7107, a small molecule that inhibits the SF3b core spliceosome complex. Treatment with either drug dramatically increased cassette exon skipping events and intron retention relative to DMSO control, with greater effects in splicing mutant cells. However, at equipotent doses, E7820 markedly increased mis-splicing compared with E7107. Furthermore, E7820 treatment resulted in mis-splicing of a number of RBP targets identified in our CRISPR screen as being required for AML survival, including SUPT6H, hnRNPH, and SRSF10, as well as RBM3 and U2AF2, consistent with previous observations (Fig. H). Here through systematic evaluation of RBPs across several cancers, we identify RBPs specifically required in AML. In so doing we identify a network of functionally and physically interacting RBPs upregulated in AML over normal precursors. Genetic or pharmacologic elimination one such RBP, RBM39, led to aberrant splicing of multiple members of this RBP network as well as of transcriptional regulators required for AML survival. These data suggest important clinical potential for anti-cancer sulfonamide treatment in splicing mutant myeloid leukemias. Disclosures Uehara: Eisai: Employment. Owa:Eisai: Employment.

Abstract The identification of mutations in BRAFV600E and other kinases in systemic histiocytoses changed our understanding of the pathophysiology of these disorders from inflammatory non-malignant conditions to clonal disorders driven by altered MAP kinase signaling. In addition, BRAFV600E mutations have recently been noted in bone marrow (BM) CD34+ cells of patients with Langerhans Cell Histiocytosis (LCH), suggesting that LCH represents a clonal disorder arising from hematopoietic stem/progenitor cells (HSPCs). However, it is unknown if HSPCs from LCH or other histiocytoses patients can functionally give rise to these disorders. Moreover, whether or not patients with histiocytosis have additional hematological disorders related to the presence of HSPC-based mutations is unclear. Here we attempted to understand the spectrum of hematological disorders in patients with histiocytoses and the cell-of-origin of histiocytic neoplasms by functionally analyzing HSPCs from histiocytoses patients. We first analyzed a multicenter cohort of 190 adult histiocytoses patients. This unexpectedly identified a high frequency of patients with a co-existing myeloid malignancy. Of the 190 patients, 9.5% (18/190) were diagnosed with Erdheim-Chester Disease (ECD) and/or LCH concomitantly with a classic MPN (3.7%; 7/190), MPN/MDS overlap (4.7%; 9/190), or MDS (1.1%; 2/190) (Figure). This clinical observation was confirmed by detection of co-existing histiocytoses-associated kinase mutations (including BRAF, MAP2K1, NRAS, or KRAS mutations) with JAK2 or CALR mutations, as well as other mutations recurrently found in myeloid malignancies (such as IDH1/2, ASXL1, and TET2 mutations (Figure)). These data therefore reveal a heretofore-unrecognized clinical overlap of adult-onset systemic histiocytoses and WHO-classified myeloid malignancies while further highlighting the possibility of an aberrant HSPC origin for histiocytic neoplasms. In most cases, the BRAF/MAP2K1/RAS mutation predominated in the histiocytic lesion while the JAK2/CALR mutation predominated in the BM/peripheral blood suggesting likely distinct clones driving each clinical condition. Next, to test the self-renewal and differentiation capacity of HSPCs from histiocytoses patients, CD34+ cells were purified from 8 patients including 6 with ECD or LCH plus a concomitant myeloid neoplasm, and 2 with ECD alone. 0.1-0.8x106 CD34+ cells from each patient were transplanted by intrafemoral injection into sublethally irradiated NOD/SCID IL2Rγ transgenic human GM-CSF, IL-3, and SCF expressing (NSG-SGM3) mice. Human engraftment was monitored by monthly flow cytometric analysis of peripheral blood until mice became moribund. Thereafter, animals were sacrificed and tissues were analyzed using flow cytometry, histology, immunohistochemistry, and sequencing analyses to detect driver mutations identified in the histiocytosis/co-occurring myeloid neoplasm. Successful engraftment of human myeloid cells was verified in grafts generated from 2/8 patients after a mean of 90 days (range: 60-120 days). This included lethal engraftment of KRAS-mutated ECD patient cells at 90 days manifested by infiltration of human hCD45+ cells co-expressing human myeloid and monocyte lineage markers hCD33 and hCD14 in the bone marrow, spleen, liver, lung, and kidney (Figure). Immunostains revealed that tissues were infiltrated by an hCD45+hCD163+hCD68+ population of foamy histiocytes, characteristic of ECD (Figure). Furthermore, genomic analysis of DNA from the engrafted animals BM and spleen revealed the same KRASG12S mutation found in the donor patients ECD lesion. Engraftment of a patient diagnosed with concomitant BRAFV600E-mutant ECD and ASXL1/TET2-mutant CMML was seen at 60 days. In this case, however, only engraftment of human CMML was detected without evidence of the BRAFV600E-mutant ECD component. This study suggests that adult histiocytic neoplasms frequently co-occur with other myeloid neoplasms. Moreover, histiocytosis disease-driver mutations are present in the CD34+ HSPCs, which we show for the first time can initiate disease that resembles human histiocytosis in a patient-derived xenograft. Importantly, however, even though the CD34+ compartment gives rise to both the histiocytic and myeloid neoplasm disease component, each disease is likely initiated by distinct precursors. Figure. Figure. Disclosures No relevant conflicts of interest to declare.