ALBERT

Description: Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Approximately 10% of mice transplanted with Tifab KO HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, and hypersensitivity to Toll-like receptor stimulation. To investigate the molecular function of TIFAB, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15. Moreover, we find that TIFAB enhances the deubiquitination of USP15 substrates, including MDM2 and histone 2B. To examine whether TIFAB directly regulates USP15 DUB activity, we performed in vitro deubiquitination assays in a cell-free system using fluorescent reporter di-ubiquitin substrates with purified USP15. We found that the addition of purified TIFAB increases the rate of USP15 catalytic activity on both lysine (K)48 and K63-linked di-ubiquitins in a dose-dependent manner. Collectively, these findings indicated that the USP15-TIFAB interaction leads to increased USP15 activity. USP15 stabilizes MDM2 via its DUB function, and MDM2 is known to bind and inhibit p53. Moreover, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21. Collectively, our findings identify a novel role for TIFAB as an activating adapter of USP15 and mediator of p53 activity. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML. Disclosures No relevant conflicts of interest to declare.

Description: SF3B1 mutations are the most frequently occurring splicing factor mutations in MDS and AML, however the misspliced genes that contribute the malignant state in SF3B mutant MDS or AML remains unclear. We determined that SF3B1 mutant cases of MDS express a longer, active isoform of interleukin 1 receptor associated kinase (IRAK4). IRAK4 is a serine/threonine kinase that is downstream of toll-like receptor (TLR) signaling and leads to activation of oncogenic signaling states, including NF-kB and MAPK. Examination of IRAK4 by RNA sequencing showed that normal cells predominantly express small IRAK4 isoforms resulting from exclusion of the part of exon 6. These isoforms are targeted for proteosomal degradation leading to diminished IRAK4 expression and activation in normal cells. In contrast, a large proportion of MDS/AML samples with SF3B1 mutation show increased expression of an IRAK4 isoform that retains full exon 6, encoding the full-length protein (IRAK4-Long). Consequently, we show that expression of mutant SF3B1-K700E in leukemic cells is associated with increased NF-kB activity, suggesting that mutations in SF3B1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Furthermore, SF3B1 mutant MDS and AML cells exhibited a block in hematopoietic differentiation in clonogenic assays. This differentiation block was ameliorated with pharmacologic inhibition of IRAK4 with CA-4948, a potent oral clinically useful small-molecule inhibitor of IRAK4. CA-4948 blocked TLR-stimulated cytokine release in various cell models and also led to decreased leukemic burden in mice xenografted with SF3B1 mutant MDS/AML cells. Finally, we determined that SF3B1 mutation induced IRAK4 activation led to TRAF6 mediated K63 ubiquitination of critical cell cycle and regulatory proteins directly implicated in oncogenesis. We had recently shown that U2AF1 mutations can lead to IRAK4 activation via retention of exon 4 (Smith et al, Nat Cell Bio, 2019). Our data now demonstrate that SF3B1 leads to overactivation of IRAK4 via retention of a different exon (exon 6), thus reinforcing that IRAK/TRAF6 activation is a common downstream oncogenic pathway in splicing factor mutated MDS/AML. Taken together, in this study, we find that mutations in SF3B1 induce expression of therapeutically targetable "active" IRAK4 isoforms and provide a genetic link between a spliceosome mutation and oncogenic innate immune signaling in MDS and AML. Disclosures Booher: Curis: Employment. Ramachandra:Aurigene: Employment. Samson:Curis: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Research Funding. Starczynowski:Kurome Therapeutics: Consultancy. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

Description: Inflammatory and innate immune signaling pathways are activated in leukemic stem and progenitor cells and contribute to the pathogenesis of hematologic malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). UBE2N is a ubiquitin (Ub) conjugating enzyme that catalyzes lysine 63 (K63)-linked Ub chains on substrates that are critical for signal transduction of broad innate immune signaling pathways. Here we report that UBE2N is required for leukemic cell function by mediating oncogenic innate immune signaling, and identified a novel chemical class of small molecule inhibitors that target UBE2N enzymatic activity. Upon UBE2N downregulation with two lentivirally expressed shRNAs, MOLM-13 and THP-1 cells lose their clonogenic potential and undergo cell death. Unlike for AML cells, UBE2N was dispensable for colony formation and viability of healthy cord blood CD34+ cells. The active site of UBE2N contains a cysteine (Cys) at position 87, which is essential for binding and transfer of Ub to its substrates. We performed in silico structure- and in vitro cell-based screens to identify small molecules that dock to the active site of UBE2N and covalently bind to Cys-87, as an approach to inhibit Ub transfer to substrates. Two structurally-related candidates, UC-764864 and UC-764865, emerged as inhibitors of UBE2N, as they specifically blocked the E1-UBE2N thioester transfer in vitro. Treatment of MDS/AML cell lines and patient-derived primary cells with UC-764864 and UC-764865 suppressed innate immune signaling and induced cytotoxic effects in MDS/AML cell lines and primary cells while sparing healthy hematopoietic cells in vitro and in vivo. To identify the molecular basis of UBE2N inhibition, we performed a global Ub screen for changes in ubiquitinated substrates by mass spectrometry and evaluated changes in gene expression by RNA-seq in MOLM-13 cells treated with vehicle control or the newly derived UBE2N inhibitors. RNA-seq of MOLM-13 cells treated with UC-764864 revealed that inhibition of UBE2N in leukemic cells targets oncogenic innate immune pathways, including NF-kB and Type I interferon signaling networks. UC-764864 and UC-764865 reduced the ubiquitination status of UBE2N, and altered the ubiquitination of proteins involved in innate immune signaling and the DNA damage response by primarily reducing K63-linked Ub modifications. Two substrates identified by the Ub screen, DDB1 and UBE2M, are components of the CUL4-CRBN E3 ligase complex and a target of the anti-leukemic therapy, Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and UBE2N inhibitors on MDS/AML cells. As compared to individual treatments, the combination of LEN and UC-764864, UC-764865 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and UBE2N inhibitors impaired MDS/AML cells that are refractory to treatment of LEN or UBE2N inhibitors alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS or AML. In summary, we implicate the Ub conjugating enzyme UBE2N as a target in MDS/AML, and identified novel small molecule inhibitors that target UBE2N and modify the function of Ub E3 ligases that are important for UBE2N-associated diseases, including autoinflammatory and autoimmune disorders, and hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem and progenitor cells (HSPC) from MDS and AML patients exhibit overexpression of TRAF6 and related innate immune pathway genes, suggesting a dependency of leukemic HSPC on activated innate immune signaling. Unfortunately, inhibiting TRAF6 directly has proven difficult, as few binding pockets on TRAF6 exist for small molecule targeting. UBE2N/Ubc13, a cofactor of TRAF6 and key enzyme in innate immune signaling, is an ubiquitin-conjugating E2 enzyme that catalyzes lysine 63 (K63)-linked ubiquitin chains on TRAF6 and its substrates. Importantly, a commercially available compound and our own chemical series of UBE2N inhibitors are available. In this study we evaluated the cellular and molecular effects of pharmacologic and genetic inhibition of UBE2N in MDS and AML cells. Pharmacologic inhibition of UBE2N with NSC697923 or genetic inhibition with shRNAs reduced the clonogenic capacity of MDSL/AML cell lines and primary cells while not significantly affecting normal HSPC. Treatment of MDS/AML cells with NSC697923 reduced the cellular metabolic activity, induced a G2/M cell cycle arrest, and increased cell death. Moreover, xenotransplantation of an MDS-derived patient cell line (MDSL) into immunodeficient mice (NSG-SGM3) showed a 50-70% reduced graft upon UBE2N knockdown relative to a non-silencing control. The cellular effects of UBE2N inhibition correspond with suppression of TRAF6-induced NF-kB activation of target genes. In addition, we found that NSC697923 treatment results in a dramatic loss of TRAF6 protein expression, which is rescued by inhibition of the proteasome. Intriguingly, our molecular analysis revealed that UBE2N inhibition shifts the stoichiometry of TRAF6 ubiquitin chains from K63-linked to K48-linked ubiquitin, resulting in proteasome-mediated degradation. To identify the molecular basis of UBE2N inhibition, we performed a global ubiquitin screen for changes in ubiquitinated substrates and gene expression profiling by RNA sequencing. For the ubiquitin screen, K63 ubiquitinated proteins were immunoprecipitated from MDSL cells upon pharmacologic inhibition of UBE2N, followed by mass spectrometry analysis. UBE2N inhibition significantly altered the ubiquitination of ~140 proteins involved in innate immune signaling, glycolysis, cell survival, RNA splicing, and DNA damage response. In parallel, RNA sequencing of MDSL cells treated with NSC697923 revealed expression changes in genes involved in mRNA processing, cell cycle and glycolysis. Several components of the E3 ligase anaphase-promoting complex APC/CDC20 were downregulated after UBE2N inhibition. As expected, increased expression of APC/CDC20 substrates (i.e., cyclin B1) were observed following treatment with NSC697923, suggesting that UBE2N inhibition in MDS/AML blocks degradation of APC/CDC20 targets and leads to mitotic alterations and apoptosis. One substrate identified in NSC697923-treated MDSL cells by the ubiquitin screen is DDB1, a component of the CUL4-CRBN E3 ligase complex targeted by Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and NSC697923 on MDS/AML cells. As compared to individual treatments, the combination of LEN and NSC697923 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and NSC697923 impaired MDS/AML cells that are refractory to treatment of LEN or NSC697923 alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS/AML. In summary, our data reveal a novel therapeutic target, an E2 ubiquitin conjugating enzyme (UBE2N), in MDS/AML. UBE2N inhibition suppresses the function and viability of MDS/AML cell lines and patient samples, due in part to degradation of TRAF6, suppressing innate immune signaling, and inducing mitotic alterations. Lastly, we show that inhibition of UBE2N alters ubiquitination of DDB1, a component of the CRBN complex, and cooperates with LEN to target MDS/AML cells. Disclosures No relevant conflicts of interest to declare.

Description: Alternative RNA splicing and mutations in spliceosome genes are common features of human cancer. For example, recurring mutations of spliceosome genes in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) most frequently involve U2AF1, SF3B1, and SRSF2. Although aberrant RNA splicing is implicated in the pathogenesis of human cancers, it is less understood which misspliced genes contribute the malignant state. A global analysis of alternatively spliced genes and RNA isoforms in breast cancer, lung cancer, and AML revealed enrichment of alternatively spliced genes associated with inflammatory and immune pathways in the cancer cells as compared to the respective normal tissues. One such example is cancer-specific isoform expression of Interleukin Receptor Associated Kinase 4 (IRAK4), a serine/threonine kinase downstream of toll-like receptor (TLR) signaling and implicated in the pathogenesis of MDS/AML (Rhyasen et al., Cancer Cell 2013). Examination of the spliced isoforms by RNA sequencing showed that normal tissues preferentially express an alternatively spliced isoform of IRAK4 resulting from exclusion of the exon 4 (IRAK4-Short). In contrast, the majority of MDS and AML samples exclusively express an IRAK4 isoform that includes exon 4 (IRAK4-Long). Skipping of IRAK4 exon 4 results in an in-frame deletion of the N-terminal death domain, which is required for IRAK4 oligomerization and efficient TLR signaling, yet retains its C-terminal kinase domain. Immunoblotting confirmed that MDS/AML samples predominantly express the IRAK4-Long protein, while normal hematopoietic BM cells express the IRAK4-Short protein lacking the N-terminal domain. IRAK4-Long expression is significantly associated with increased NF-kB and innate immune signaling and correlates with poor AML patient outcome. Functional characterization of the IRAK4 isoforms in human AML cell lines revealed that IRAK4-Long induces NF-kB activation. In contrast, IRAK4-Short is less efficient at activating NF-kB (via phosphorylation of IKKbeta), yet it activates p38/MAPK signaling. To gain insight into the alternative splicing regulation of IRAK4 exon 4, we examined IRAK4 isoform expression and associated spliceosome gene mutations in MDS/AML patients. Of all examined genetic associations, mutation of U2AF1 (S34F) significantly correlated with inclusion of exon 4 and expression of IRAK4-Long, suggesting that mutations in U2AF1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. To explore the direct regulation of IRAK4 by U2AF1, wildtype or mutant (S34F) U2AF1 were expressed in CD34+ cord blood cells, and IRAK4 isoform expression and exon usage was determined by RNA-sequencing. Expression of U2AF1-S34F resulted in significant retention of IRAK4 exon 4 (i.e. IRAK4-Long), while expression of wildtype U2AF1 correlated with exclusion of IRAK4 exon 4 (i.e., IRAK4-Short). Moreover, exon 4 and flanking intron sequences were cloned into a splicing reporter. Overexpression of U2AF1-S34F induced retention of the cassette exon 4, while wild-type U2AF1 mediated exclusion of exon 4. Taken together, these findings illustrate the importance of cancer-associated RNA splicing alterations and their consequences on downstream molecular networks required for cancer pathogenesis. In addition, we find that U2AF1 mutations result in expression of IRAK4 isoforms providing a genetic link to chronic innate immune signaling and IRAK1/4 activation in MDS and AML. Disclosures No relevant conflicts of interest to declare.

Description: Alternative RNA splicing and mutations in spliceosome genes are common features of MDS and AML, however which misspliced genes contribute the malignant state remains unclear. A global analysis of intron and exon usage in AML revealed enrichment of alternatively spliced genes associated with inflammatory and immune pathways in leukemic cells as compared to normal cells. The gene with the greatest differential isoform expression was IRAK4, a serine/threonine kinase downstream of toll-like receptor (TLR) signaling. Examination of IRAK4 by RNA sequencing showed that normal cells preferentially express an IRAK4 isoform resulting from exclusion of the exon 4, which encodes a protein lacking the N-terminal death domain (IRAK4-Short). In contrast, a large proportion of MDS/AML samples show increased expression of an IRAK4 isoform that retains exon 4, encoding the full-length protein (IRAK4-Long). Of all somatic genetic mutations associated with AML, mutation of U2AF1 (S34F) significantly correlated with inclusion of exon 4 and expression of IRAK4-Long. Examination of IRAK4 exon 4 usage in CD34+ cells from genetically-defined MDS patient samples revealed that nearly all MDS patient samples containing mutations in U2AF1 exhibited increased inclusion of exon 4 as compared to WT U2AF1 MDS samples or healthy controls. Strikingly, SF3B1 mutant MDS patient samples also exhibited increased expression of IRAK4-Long, suggesting a unifying mechanism of IRAK4 isoform expression in spliceosome mutant MDS/AML. Higher expression of IRAK4-Long in MDS CD34+ cells or AML blasts is associated with poor prognosis, and correlates with elevated blast counts and transfusion dependency in MDS. Utilizing a splicing reporter containing exon 4 and flanking intron sequences of IRAK4, U2AF1-S34F induced retention of the cassette exon 4, while WT U2AF1 mediated exclusion of exon 4. Ectopic expression of U2AF1-S34F in AML cells resulted in significant retention of IRAK4 exon 4 and expression of IRAK4-Long protein. Functional characterization of the IRAK4 isoforms in human AML cell lines revealed that IRAK4-Long induces NF-kB activation, suggesting that mutations in U2AF1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Importantly, U2AF1-S34F AML cells were more sensitive to pharmacologic inhibition of IRAK1/4 as compared to isogenic cells with WT U2AF1. CA-4948, a potent oral small-molecule inhibitor of IRAK4, blocked IRAK4-mediated signaling in TLR-stimulated THP1 AML cells, and decreased leukemic burden in xenografted mice. Finally, CA-4948 treatment led to reduced leukemic engraftment of primary patient MDS/AML samples with expression of IRAK4-Long. Taken together, we find that mutations in U2AF1 and SF3B1 induce expression oftherapeutically targetable"active" IRAK4 isoforms and provide a genetic link to chronic innate immune signaling and IRAK1/4 activation in MDS and AML. Disclosures Steidl: Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding. Booher: Curis, Inc: Employment, Equity Ownership.