ALBERT

Description: Myelodysplastic syndromes (MDS) are defined by blood cytopenias due to ineffective hematopoiesis, and predisposition to acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic alteration in MDS is deletion of chromosome 5q (del(5q)). A search of annotated genes within or near the commonly-deleted regions (CDR) in del(5q) revealed a novel uncharacterized gene, TRAF-interacting protein with forkhead-associated domain B (TIFAB). TIFAB resides within the proximal CDR on band 5q31.1, and consistent with haploinsufficiency, expression of TIFAB mRNA is at least 50% lower in del(5q) MDS as compared to diploid 5q MDS and age-matched control marrow cells. Restoring TIFAB in human AML cell lines with low endogenous levels of TIFAB results in increased apoptosis, diminished proliferation, and impaired leukemic progenitor function, suggesting that it functions as a tumor suppressor. To investigate whether loss of TIFAB is important to the pathophysiology of del(5q) MDS/AML, we characterized a novel germline TIFAB knockout (KO) mouse for hematopoietic stem/progenitor cell (HSPC) function. Whole-body TIFAB KO mice do not exhibit changes in steady-state hematopoiesis even beyond 1 year of age. However, the consequences of 5-fluorouracil (5-FU) treatment on TIFAB KO mice were more severe as compared to wild type (WT)-treated mice, suggesting that HSPC function of TIFAB KO mice is affected following hematopoietic stress. To further investigate the consequences of TIFAB loss on HSPC function, TIFAB KO marrow cells were transplanted into WT syngeneic recipient mice. For 3 months post-BM transplant, WT and TIFAB KO-transplanted mice displayed similar blood and BM proportions. However, beyond 6 months post-BM transplantation, TIFAB KO mice displayed progressive hematopoietic defects, including skewed HSPC proportions, altered myeloid differentiation, and pancytopenia. Importantly, approximately 40% of mice transplanted with TIFAB KO BM cells developed a BMF-like disease associated with BM dysplasia and pancytopenia. Cell-intrinsic HSC defects in TIFAB KO mice were confirmed by performing competitive BM transplantations. In support of a HSC defect, TIFAB KO HSC were out-competed by co-transplanted WT HSC. To uncover the molecular consequences that explain the HSPC defects in TIFAB KO mice, we isolated Lin-cKit+Sca1+ (LSK) and performed a microarray analysis. TIFAB KO LSK exhibited an increase in HSC-, IFN-, and p53-related gene signatures and downregulation of LPS-stimulated gene signatures as compared to WT LSK. Despite a repression of an LPS-stimulated gene signature, TIFAB KO BM cells are hyper-sensitive to LPS stimulation, suggesting that loss of TIFAB alters the innate immune pathway. As a potential explanation for altered LPS sensitivity, TIFAB loss induces an increase in TRAF6 protein, a key modulator of the innate immune pathway. Taken together, our results provide evidence that TIFAB exhibits tumor suppressor-like functions and that deletion of TIFAB contributes to an MDS-like phenotype in mice by changing the dynamic range of the innate immune pathway reactivity in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

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: Myelodysplastic Syndromes (MDS) often progress to Acute Myeloid leukemia (AML) typically following acquisition of mutations in RUNX1, NRAS/KRAS or AXSL1. Inflammatory processes associated with autoimmune diseases or natural aging are thought to contribute to the development of MDS and progression to AML. Moreover, dysregulation of innate immune signaling is broadly observed in hematopoietic stem and progenitor cells (HSPC) from MDS patients. However, how dysregulation of innate immune signaling and/or inflammatory signals might lead to malignant transformation and overt AML has not been fully elucidated. We have developed a mouse model of cell-intrinsic inflammation-driven progression from MDS to AML that will allow us to determine the mechanisms of oncogenic transformation. MicroRNA miR-146a is a 5q gene that is frequently deleted in MDS and AML and its deletion promotes dysregulation of innate immune signaling and a systemic inflammatory state. Deletion of miR-146a results in myeloproliferation and marrow failure in mice associated with attrition of HSPC, but alone is insufficient to induce overt AML. Using a genetically engineered approach, we show that deficiency of miR-146a (miR-146a-/-) combined with a C-terminal truncation of the RUNX1 (S291fsX300) protein causes anemia, thrombocytopenia and ineffective erythropoiesis mimicking human MDS in younger mice, but then the mice progresses to a fatal AML. The inflammatory environment induced by miR-146a deletion was exacerbated in mice with miR-146a-/-expressing RUNX1-S291fsX300 (RUNX1-mut) and correlated with the increased incidence and aggressiveness of hematologic malignancies. In particular, the levels of IFNγ were increased in plasma in mice with miR-146a-/-expressing RUNX1-mut in comparison to mice reconstituted with miR-146a-/-or RUNX1-mut-expressing BM cells. As previously reported, miR-146a-/-cells exhausted during serial transplantation, however RUNX1-mut rescued the hematopoietic repopulation deficiency of miR-146a-/-HSPC, suggesting that the RUNX1 mutation promotes malignant transformation to AML under inflammatory conditions. To establish whether cell-intrinsic immune signaling is required for RUNX1-mut;miR-146a-/- leukemic cells, we evaluated an inhibitor (NSC697923) that targets TRAF6, a key target of miR-146a in MDS/AML, via inhibition of its co-factor UBE2N. Treatment with NSC697923 or a RUNX1 inhibitor (Ro 5-3335) abolished colony formation by RUNX1-mut;miR-146a-/- leukemic cells, indicating that cell-intrinsic innate immune signaling is not only required for initiating AML, but is also critical for sustaining the leukemic phenotype. These findings describe the first evidence that dysregulation of innate immune signaling and/or inflammatory signals contribute to the progression and maintenance of AML. Disclosures Starczynowski: Kurome Therapeutics: Consultancy.

Description: Toll-like receptors (TLR) are known for regulating myeloid homeostasis and response to infection, but chronic activation of TLR pathways can also lead to hematopoietic stem and progenitor cell (HSPC) dysfunction. Furthermore, mutations that lead to constitutive activation of TLR pathways contribute to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS); however, the underlying cellular and molecular mechanisms are unknown. As a means of chronically activating TLR signaling within HSPCs, we generated a mouse model by elevating expression of TRAF6 in hematopoietic cells (Vav-TRAF6). TRAF6 is a downstream TLR-effector with ubiquitin (Ub) E3 ligase activity, and is overexpressed in MDS HSPCs. Vav-TRAF6 mice developed progressive leukopenia and anemia, and exhibited myeloid skewing and dysplasia. Eventually, over half of Vav-TRAF6 mice succumbed to a bone marrow (BM) failure associated with MDS. Despite increased frequencies of immunophenotypic HSPCs in the BM, Vav-TRAF6 HSPCs are functionally defective as evidenced by impaired colony formation and reduced in vivo competitiveness. The hematopoietic phenotype due to TRAF6 overexpression was still manifest upon transplantation, indicating that the effect is hematopoietic cell intrinsic. Consistent with the cellular effects observed with chronic TLR activation, elevated TRAF6 expression results in MDS/BMF by altering intrinsic HSPC properties. Gene expression and exon level analyses revealed that Vav-TRAF6 HSPCs exhibit discrete and durable alterations in RNA splicing patterns. The family of small G-protein GTPases emerged as a relevant pathway whose activity is affected by missplicing of Arhgap1, a GTPase activating protein. Aberrant skipping of Arhgap1 exon 2 resulted in reduced Arhgap1 protein and constitutive Cdc42 GTPase activation. Inhibition of Cdc42 activity with a pharmacological inhibitor partially reversed myeloid-biased differentiation of Vav-TRAF6 HSPCs in vivo, indicating that missplicing of Arhgap1 and increased Cdc42 activity accounts for several HSPC defects. To identify the mechanism underlying TRAF6-induced RNA splicing, we employed a global Ub-enrichment screen for novel TRAF6 substrates, and uncovered hnRNPA1, an RNA-binding protein that regulates exon usage. hnRNPA1 is ubiquitinated by TRAF6 adjacent to and within its first RNA-binding domain. hnRNPA1 binding sites were significantly enriched within misspliced exons in Vav-TRAF6 HSPCs and in primary human AML samples with elevated TRAF6 expression, indicating that TRAF6 overexpression induces exon skipping via hnRNPA1. The requirement of hnRNPA1 in TRAF6-induced exon skipping was confirmed as knockdown of hnRNPA1 significantly reduced Arhgap1 exon 2 skipping in Vav-TRAF6 HSPC. Moreover, depletion of hnRNPA1 reversed Vav-TRAF6 hematopoietic defects in vivo, unequivocally validating the importance of hnRNPA1 in TRAF6-mediated exon skipping and function of HSPCs. Our findings uncover a novel mechanism by which sustained TLR signaling, via TRAF6-mediated ubiquitination of hnRNPA1, alters RNA splicing and contributes to MDS-associated HSPC defects in part by activating Cdc42. These results indicate a novel function for Ub signaling in coordinating transcriptional initiation and alternative splicing by TLR signaling pathway within the immune system and in premalignant hematologic diseases, such as MDS. Disclosures Starczynowski: Celgene Corporation: Research Funding.

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: Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function. To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis. To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells). Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. Disclosures No relevant conflicts of interest to declare.