ALBERT

Description: Overexpression of immune-related genes is widely reported in Myelodysplastic Syndrome (MDS), and chronic immune stimulation increases the risk for developing MDS. We find that TNF receptor associated factor 6 (TRAF6), an innate immune protein, is overexpressed approximately 2-fold in CD34+ cells from 40% of MDS patients, and may explain immune pathway activation in the MDS-initiating hematopoietic stem/progenitor cell (HSPC). In support of these observations and our hypothesis that TRAF6 is important in the pathophysiology of MDS, a gene expression analysis revealed that TRAF6 controls an MDS gene signature in human cells. We, and others, have previously shown that retroviral overexpression of TRAF6 in mouse HSPC results in MDS and Acute Myeloid Leukemia (AML). However, interpretations of these findings are hampered by supra-physiological levels of TRAF6 (〉10-fold overexpression) and the stress associated with HSPC transduction/transplantation. To investigate the consequences of TRAF6 overexpression to MDS, we generated a transgenic mouse model overexpressing TRAF6 from a hematopoietic-specific Vav promoter. Expression of TRAF6 in HSPC was approximately 2-fold higher as compared to endogenous TRAF6 and in line with MDS patient CD34+ cells. By 15 months of age, half of Vav-TRAF6 mice succumbed to a hematologic disease resembling MDS associated with bone marrow failure (BMF). In contrast to the retroviral overexpression approach, Vav-TRAF6 mice did not develop AML. Examination of sick mice revealed stage-specific disease evolution. Initially, all Vav-TRAF6 mice exhibit an inversion of myeloid/lymphoid proportions. For Vav-TRAF6 mice that develop a fatal disease, they present with a hypocellular marrow, dysplasic myeloid cells, and neutropenia. A subset of mice also display anemia with nucleated red blood cells, poikilocytosis, and extramedullular erythropoiesis. In support of a BMF phenotype, HSPC from Vav-TRAF6 mice form fewer colonies in methylcellulose. To investigate the consequences of an acute exposure to pathogen, early-stage Vav-TRAF6 mice were treated with a single sublethal dose of lipopolysaccharide (LPS). Unlike wild-type (WT) mice, Vav-TRAF6 mice developed a rapid and reversible anemia, suggesting environmental factors can influence the severity of the disease. To gain insight into the mechanism contributing to BMF, gene expression profiling was performed in WT and Vav-TRAF6 HSPC. One of the enriched pathways consisted of AKT activation and FOXO downregulation. Consistent with the microarray analysis, AKT is constitutively phosphorylated at Thr308 in hematopoietic tissue from Vav-TRAF6 mice. SOD2, a transcriptional target of FoxO3a that is suppressed by activated AKT, is decreased in Vav-TRAF6 HSPC. Given that AKT/FOXO regulate reactive oxygen species (ROS) in cells, we investigated ROS levels in HPSC from Vav-TRAF6 and WT mice. Intracellular ROS is significantly elevated in BM cells from Vav-TRAF6 mice, and restored to normal levels when AKT was inhibited. In conclusion, we propose the potential role of TRAF6 in the development of MDS-associated BMF, partly due to constitutive activation of AKT and subsequent ROS elevation in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 557 Deletion of chromosome 5q (del(5q)) is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Commonly deleted regions (CDR) have been mapped to 5q31.1 and 5q33.1 in del(5q) AML and MDS, respectively. Although there has been extensive efforts to identify candidate genes within the CDRs and decipher which genes contribute to the high-risk versus low-risk phenotypes, more recent findings indicate that deletions involving the telomeric bands of 5q are associated with more aggressive forms of del(5q) MDS/AML (Jerez et al., JCO, 2012). To determine which genes on the telomeric extremes of chr 5q contribute to the aggressive phenotype associated with these patients, we performed a gene expression and functional analysis of candidate tumor suppressor (TS) genes located between ∼5q34 to 5q35.3. Among the 7 candidate TS genes localized on the telomeric extreme, expression of miR-146a is significantly reduced in del(5q) MDS/AML marrow cells with extended deletions as compared to ones with shorter deletions. In addition, low expression of miR-146a correlates with increased marrow blasts and shorter overall survival of patients with del(5q). To investigate the effects of miR-146a on hematopoietic stem/progenitor cell (HSPC) function, we stably knocked down miR-146a in human CD34+ cord blood and mouse HSPC-enriched cells. HSPC cells with reduced miR-146a exhibited features of malignant transformation, including increased survival, proliferation, and colony replating in methylcellulose. The mechanism underlying reduced miR-146a expression was investigated by performing gene expression profiling on marrow cells from MDS/AML patients with extended and short del(5q). Gene Ontology and Gene Set Enrichment Analysis revealed that patients with low expression of miR-146a exhibited a significant increase in cell cycle, immune response, and NF-kB target genes. These findings are consistent with the model that reduced levels of miR-146a results in derepression of TRAF6, a signaling protein within the innate immune pathway and activator of NF-kB (Starczynowski et al., Nature Medicine 2010). TRAF6 forms a signaling complex with Sequestosome 1 (SQSTM1/p62), a gene located within the telomeric deleted region in del(5q) on band 5q34. Surprisingly, expression of p62 was not reduced, but rather overexpressed in del(5q) MDS/AML patients with the extended deletions and inversely correlated with miR-146a expression. The compensatory expression of p62 is due in part by loss of miR-146a. RNAi-mediated knockdown revealed that p62 and TRAF6 are essential for NF-kB activation and survival of human MDS/AML cell lines with low miR-146a expression. In summary, we identified that miR-146a is a TS-like gene in MDS/AML patients with extended deletions of chr 5q, and that reduced miR-146a expression increases HSPC survival/proliferation and NF-kB signaling via p62 and TRAF6. Therefore, we propose that inhibiting the p62/TRAF6/NFκB intrachromosomal gene network represents a novel targeting strategy in high-risk MDS/AML patients with extended chr 5q deletions. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Description: Deletions of the long arm of chromosome 5 (del(5q)) are common cytogenetic alterations in myelodysplastic syndrome (MDS), a disease characterized by refractory anemia, megakaryocyte dysplasia, and thrombocytosis. The thalidomide analogue lenalidomide (LEN) produces durable erythroid responses in ~60% of del(5q) MDS patients, including a majority of cytogenetic responses in which the del(5q) clone becomes undetectable in the bone marrow. Despite high response rates, clinical and cytogenetic relapse occur within 2-3 years. Mechanisms of clinical response, resistance and relapse with LEN therapy remain to be elucidated. The target of LEN has recently been identified as the cereblon (CRBN) component of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN). Upon LEN binding, the substrate-specificity of the CRL4-CRBN complex is altered, and LEN-regulated substrates are beginning to be identified. An RNA interference screen was performed to identify genes/pathways that mediate LEN sensitivity and resistance in del(5q) MDS. The LEN-sensitive del(5q) MDS patient-derived cell line MDSL was screened with a genome-wide shRNA library (SBI GeneNet Human 50K Library) in the presence and absence of LEN treatment (0 and 10 μM) for 7 days. Three independent shRNAs targeting the proton-sensing G protein-coupled receptor 1 (GPR68 or OGR1) were among the most enriched shRNAs in LEN-treated cells, suggesting that loss of GPR68 expression conferred resistance to LEN. This finding was validated in MDSL cells, using an independent set of shRNAs. Conversely, a GPR68 agonist (N-cyclopropoyl-5-[thiophen-2-yl]-isoxazole-3-carboxamid) enhanced LEN-induced cytotoxicity to MDSL cells. GPR68 is a proton-sensing G-protein coupled receptor that stimulates inositol phosphate production and/or intracellular calcium (Ca2+) mobilization. Curiously, CRBN was originally identified as a binding protein of calcium-activated potassium channels. These data led us to hypothesize that Ca2+ signaling may be responsible for LEN-mediated cytotoxic effect in MDS cells. Reducing intracellular Ca2+ level with chelators reversed LEN’s cytotoxic effects, while increasing intracellular Ca2+ level with ionomycin enhanced LEN’s cytotoxic effect, indicating that intracellular Ca2+ levels determine cellular responsiveness to LEN. Although LEN did not induce an instant burst of Ca2+ influx, a gradual increase of basal intracellular free Ca2+ was observed following LEN treatment in LEN-sensitive cell lines and primary MDS marrow cells, but not in LEN-resistant cells, suggesting that LEN cytotoxicity was dependent on the cell’s ability to release Ca2+ from intracellular stores. GPR68 and CRBN were both necessary for the LEN-induced increase in Ca2+, as knockdown of GPR68 or CRBN in LEN-sensitive cells prevented the Ca2+increase. To identify the Ca2+-dependent signaling pathway responsible for mediating the cytotoxic effect of LEN, a panel of seven inhibitors that blocked mitochondrial/caspase-, calpain-, autophagy-, or lysosomal-dependent cell death pathways was tested in combination with LEN on MDSL cells. Only the inhibitor of calpains (PD150606) prevented LEN-induced cytotoxic effects in MDSL cells, indicating that calpain activation was necessary for mediating cell death in LEN-treated cells. Calpains are Ca2+-dependent cysteine proteases that can induce apoptotic and necrotic cell death by proteolytic cleavage of protein substrates. Calpastatin, the only endogenous calpain inhibitor, is localized to 5q15 and its expression is haploinsufficient in del(5q) MDS as compared to normal karyotype MDS. Taken together, our results show that LEN increased intracellular Ca2+ levels by a CRBN- and GPR68-dependent mechanism, leading to calpain-mediated cytotoxicity in del(5q) MDS cells. We propose a model in which haploinsufficient expression of calpastatin in del(5q) MDS sensitizes cells to cytotoxic effects of LEN. Further studies are required to identify the direct LEN-modulated substrates of CRBN that mediate this effect. Disclosures Oliva: Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria. MacBeth:Celgene: Employment, Equity Ownership. Starczynowski:Celgene: Research Funding.

Description: Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

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: Abstract 2812 Myelodysplastic syndromes (MDS) are hematologic disorders defined by blood cytopenias due to ineffective hematopoiesis, altered cytogenetics, and predisposition to acute myeloid leukemia (AML). The most common cytogenetic alteration in de novo and treatment-related MDS is deletion of chromosome 5q (del(5q)). There are two commonly deleted regions (CDR) mapped to chr 5q, however the gene(s) in these regions responsible for the manifestation of del(5q) MDS are not clearly defined. A search of annotated genes revealed that TRAF-interacting protein with forkhead-associated domain B (TIFAB), a known inhibitor of TRAF6 and a novel gene identified by an in silico search for TIFA-related genes, resides within the proximal CDR on band 5q31.1. We first determined whether TIFAB is expressed in normal hematopoietic stem/progenitor cell (HSPC) by qRT-PCR. We find that expression of TIFAB is enriched in human CD34+/CD38+ and mouse lineage-/cKit+ progenitors as compared to more differentiated populations, suggesting that it plays a role in normal HSPC function. To determine whether TIFAB is implicated in del(5q) MDS, we measured TIFAB expression in del(5q) MDS patients. According to a microarray analysis, TIFAB mRNA was significantly lower in CD34+cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q (Pellagatti, et al., 2006). In an independent subset of patients, we confirmed that TIFAB expression was lower in marrow cells isolated from del(5q) MDS patients. Therefore, we hypothesize that TIFAB loss results in hematopoietic defects contributing to del(5q) MDS. To determine whether deletion of TIFAB affects hematopoiesis, we used lentiviral shRNAs to knockdown TIFAB mRNA in human cord blood CD34+ cells. To mimic haploinsufficiency of TIFAB in del(5q) MDS, we selected shRNAs that result in ∼50% knockdown of TIFAB mRNA and protein. Knockdown of TIFAB in human CD34+ cells results in increased survival, a competitive growth advantage, and altered hematopoietic progenitor function. Conversely, overexpression of TIFAB in human leukemia cell lines (THP1 and HL60) results in increased basal apoptosis, delayed G1/S-phase cell cycle progression, and impaired leukemic progenitor function in methylcellulose. Since TIFAB is predicted to regulate TRAF6, we examined the role of TIFAB on TRAF6 signaling. TIFAB suppressed TRAF6 lysine (K)-63 autoubiquitination (a measure of TRAF6 activity), and decreased total TRAF6 protein levels, suggesting that TIFAB may simultaneously inhibit TRAF6 function and protein expression. Consistent with this finding, TIFAB suppressed lipopolysaccharide-induced (TRAF6-dependent) NF-kB activation, but not TNF-induced (TRAF6-independent) NF-kB activation. TIFAB-mediated inhibition of TRAF6 also coincided with reduced phospho-IKK-beta (a measure of NF-kB activation) in leukemic cells. In summary, we have identified TIFAB as a novel del(5q) MDS/AML gene involved in regulating HSPC survival, progenitor function, and cell cycle. We propose that haploinsufficiency of TIFAB results in malignant clonal cell expansion and may contribute to the MDS/AML phenotype as a consequence of increased TRAF6-mediated activation of NF-kB. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.