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: 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: 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: Recent studies implicate metabolic plasticity in fine-tuning hematopoietic stem cell (HSC) homeostasis and function. HSC have been shown to rely on anaerobic glycolysis due to low energetic demand. In contrast, mitochondrial respiration is largely engaged to meet high energetic demand under certain pathophysiological processes, such as differentiation, aging and malignant transformation. The microenvironment, i.e. the HSC niche, has long been known as hypoxic. Glycolytic pathway is not only the metabolic adaptation to the hypoxic niche, but also essential for HSC function at least by providing prosurvival signals. However, glycolysis is reduced in HSC upon aging, which is associated with diminished regeneration ability of HSC. How glycolysis impacts HSC biology is unclear. G protein-coupled receptor 68 (GPR68, also known as OGR1) responds to extracellular acidosis, activating the phospholipase C (PLCb)/calcium pathway or the adenylyl cyclase (CA)/cAMP pathway. The end point product of glycolysis is lactate that leads to extracellular acidosis. This prompts us to examine the role of Gpr68 in HSC biology. Among the hematopoietic stem and progenitor cell (HSPC) subpopulations, we found that Gpr68 expression was most enriched in mouse long-term HSC (LT-HSC, Lin-Sca-1+c-Kit+CD34-CD135-). To understand the function of Gpr68 in HSC biology, we generated a genetic loss of function mouse model (i.e. the Gpr68fl/fl;VavCre+ mice) where Gpr68 was specifically deleted in hematopoietic cells, including the most primitive LT-HSC. We found that the number of LT-HSC was unaltered in bone marrow (BM) from Gpr68fl/fl;VavCre+ mice as compared to wild type (WT) control littermates (i.e. the Gpr68wt/wt;VavCre+ mice), indicating that Gpr68 was not required for HSC homeostasis. To examine the role of Gpr68 in HSC function, we performed competitive bone marrow transplantation (cBMT). The donor-derived chimerism (i.e. the frequency of CD45.2+ cells) was increased in recipients with BM cells from Gpr68fl/fl;VavCre+ mice (12-month old) compared to those from age-matched WT mice after primary cBMT, which was increased even more dramatically after secondary cBMT. This indicates enhanced HSC regeneration ability due to loss of Gpr68. Increased chimerism was observed in CD3+ T lymphocytes, B220+ B lymphocytes and CD11b+ myeloid cells, indicating that both lymphoid and myeloid lineages contributed to enhanced HSC function. Consistently, increased chimerism was also observed in BM HSPC subpopulations except the LT-HSC. Of note, increased chimerism was not observed in recipients that were transplanted with BM cells from young Gpr68fl/fl;VavCre+ mice (2-month old). In addition, BM cells from Gpr68fl/fl;VavCre+ mice produced slightly reduced colonies in methylcellulose, indicating that loss of Gpr68 did not lead to malignant transformation despite enhanced competitiveness of HSC. To further understand the mechanism of Gpr68 in regulating HSC function, we studied cellular features of HSPC subpopulations. The frequency of cells in cell cycle G0 phase was unchanged in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, indicating loss of quiescence may not contribute to enhanced HSC function. Intriguingly, the frequency of Annexin V+ cells was largely reduced in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, suggesting that loss of Gpr68 provides a pro-survival signal in HSC. We next examined two main signaling pathways downstream of Gpr68. Cytosolic calcium levels were reduced in LT-HSC from Gpr68fl/fl;VavCre+ mice as compared to WT mice, indicating Gpr68 may activate a calcium-related proapoptotic pathway. In contrast, intracellular cAMP levels were increased in HSPC, possibly from unknown compensatory mechanism. In addition, Gpr68 expression was reduced in LT-HSC from older mice (12-month old) compared to younger mice (2-month old), indicating reduced Gpr68 function at 12-month of age. This is in line with reduced glycolysis in aged HSC. Our study suggests that Gpr68 mediates a proapoptotic pathway in LT-HSC, providing a negative regulation, possibly at downstream of glycolysis, that limits HSC function. Our study also suggests that inhibiting Gpr68 function would enhance HSC function, especially in aged people or during bone marrow transplantation. 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.

Description: Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML. Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function. In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway. In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cells (HSC) mainly engage glycolysis while leukemia stem cells (LSC), such as in acute myeloid leukemia (AML), heavily rely on mitochondrial (Mt) respiration (i.e. oxidative phosphorylation, OxPhos) to fuel energy. Growing evidence suggests this metabolic reprogramming confers therapeutic vulnerabilities in AML. BCL2 is overexpressed in LSC from AML patients, while BCL2 inhibitors, such as venetoclax (VEN), have been shown to suppress OxPhos in LSC and to eradicate LSC. In clinical practice, half of the patients fail to respond to VEN. VEN is a costly medication and patients who are resistant to VEN forgo alternative treatment at that time. Prediction of the response to VEN and strategies to circumvent resistance are urgently needed. BCL2 is shown to increase Mt Ca2+ levels ([Ca2+]m), which enhance OxPhos through activation of the Ca2+-sensitive dehydrogenases within the tricarboxylic acid (TCA) cycle. G protein-coupled receptor 68 (GPR68) is a proton sensor, activating phospholipase C that leads to releasing of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and elevation of cytosolic Ca2+ levels ([Ca2+]c). This prompted us to examine the cooperative effect of GPR68 and BCL2 on the Ca2+/OxPhos pathway in AML, and particularly in LSC. Expression of leukemic oncogenes (i.e. MLL-AF9 and HRASG12D) in mouse hematopoietic stem and progenitor cells, such as Lineage-Sca-1+cKit+ (LSK) cells or granulocyte-monocyte progenitor cells, promote leukemogenesis as evidenced by serial colony formation in vitro and leukemia development in vivo. We found significantly reduced colonies in oncogene-expressing LSK cells from Gpr68 knockout mice compared to wild type mice. Deletion of Gpr68 reduced [Ca2+]c in oncogene-expressing LSK cells. We next examined the function of GPR68 in human AML cell lines. Knockdown of GPR68 with shRNA reduced cell growth and colony formation, and induced apoptosis in AML cells. Knockdown of GPR68 also reduced [Ca2+]c and Mt membrane potential (Δψm) in AML cells, indicating reduced Mt OxPhos. These results suggest that GPR68 regulates the Ca2+/OxPhos pathway in AML cells. We next examined the cooperative effect of GPR68 and BCL2 by jointly inhibiting their activities with pharmacological agents (i.e. GPR68 antibody and VEN, respectively) in AML cell lines and AML patient-derived xenograft models. Of note, the expression of GPR68 was positively correlated with the sensitivity to VEN in AML cells. For AML cells that were resistant to VEN, GPR68 antibody but not an unrelated antibody increased the sensitivity to VEN by enhancing apoptosis, indicating that GPR68 and BCL2 co-regulate AML cell survival. We next examined the mechanism of this synthetic lethality by measuring cellular respiration. Single treatment with VEN reduced Δψm, ATP production and O2 consumption in AML cells. Cotreatment with VEN and GPR68 antibody further reduced Δψm, ATP production and O2 consumption in AML cells, indicating that GPR68 and BCL2 co-regulate Mt OxPhos. Given that GPR68 releases Ca2+ from ER to cytosol, while BCL2 maintains [Ca2+]m by inhibiting its extrusion from Mt, we hypothesize that the GPR68/BCL2 axis relocates Ca2+ from ER to Mt. As expected, treatment with GPR68 antibody reduced [Ca2+]c. Cotreatment with VEN and GPR68 antibody increased [Ca2+]c, indicating enhanced extrusion of Ca2+ from Mt to cytosol by VEN. Consistently, cotreatment with VEN and GPR68 antibody reduced the activity of isocitrate dehydrogenase, the rate limiting enzyme in the TCA cycle, in AML cells. These results indicate that GPR68 and BCL2 co-regulate the Ca2+/OxPhos pathway in AML cells and that co-inhibition of GPR68 and BCL2 may enhance lethality and overcome VEN resistance. In summary, our study suggests that the GPR68/BCL2 axis co-regulates AML cell survival by relocating Ca2+ from ER to Mt thus enhancing Mt OxPhos, and that disruption of the GPR68/BCL2 axis provides a novel therapeutic strategy to overcome resistance to VEN. Disclosures No relevant conflicts of interest to declare.

Description: Bortezomib (Velcade) is used widely for the treatment of various human cancers; however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that bortezomib induces proteasome-independent degradation of the TRAF6 protein, but not mRNA, in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of bortezomib due to down-regulation of the proteasomal subunit α-1 (PSMA1). To determine the molecular consequences of loss of TRAF6 in MDS/AML cells, in the present study, we applied gene-expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe herein novel mechanisms by which TRAF6 is regulated through bortezomib/autophagy–mediated degradation and by which it alters MDS/AML sensitivity to bortezomib by controlling PSMA1 expression.

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.