ALBERT

Description: Inactivation of the retinoblastoma gene (RB1) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB’s ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1+/− mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1+/− mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1-null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

Description: LBA-4 Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities. In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. Disclosures: Kaelin: Fibrogen: Consultancy, Equity Ownership.

Description: Somatic mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, are found in up to 40% of patients with myeloproliferative neoplasms (MPN). All pathologic CALR mutations are out-of-frame insertion and/or deletions (indels) in exon 9, generating a 1 base-pair (bp) frame shift and a common mutant-specific C-terminus, with the most common mutation being a 52 bp deletion (del52). The observation that CALR mutations are mutually exclusive with other MPN-initiating mutations such as JAK2V617F suggests a key pathogenic role for mutant CALR. To determine if mutant CALR alone is sufficient to induce MPN we began by over-expressing CALR-del52 in a retroviral bone marrow transplant (BMT) mouse model. We found that CALR-del52-expressing mice develop thrombocytosis and megakaryocytic hyperplasia, recapitulating the megakaryocyte-specific phenotype of CALR-mutant MPN patients. These findings suggest that the thrombopoietin receptor, MPL plays a key role in the pathogenesis of mutant CALR-driven MPN. To evaluate the role of MPL in mutant CALR driven oncogenesis, we over-expressed CALR-del52 in interleukin-3 (IL-3)-dependent Ba/F3 hematopoietic cells. We found that CALR-del52 over-expression results in transformation to IL3-independent growth only in Ba/F3 cells co-expressing MPL, but not in parental Ba/F3 cells or Ba/F3 cells co-expressing the EPO receptor (EPOR) or the G-CSF receptor (GCSFR). We found similar results in human cytokine-dependent UT-7 cells. We also introduced +1 frameshift mutations into the endogenous Calr locus in Ba/F3-MPL cells using CRISPR/Cas9 gene editing and successfully engendered IL-3 independent growth, indicating that endogenous levels of mutant Calr expression are sufficient for transformation. Together, these data indicate that MPL is specifically required for the transforming capacity of mutant CALR. Using RNA-sequencing followed by gene set enrichment analysis (GSEA), we confirmed that mutant CALR transformed Ba/F3-MPL cells display strong enrichment of Stat5 and Stat3 gene expression signatures. Concordantly, we also saw differential phosphorylation of Stat5 and Stat3 in these cells. Furthermore, we found that the IL-3 independent proliferation of mutant CALR expressing Ba/F3-MPL cells is decreased upon shRNA-mediated knockdown of Jak2, and that differential activation of Stat5 and Stat3 is abrogated by the JAK2 inhibitor, ruxolitinib. Together, these data demonstrate that mutant CALR signals through the JAK/STAT axis downstream of MPL. We next sought to define the specific domains within mutant CALR required for oncogenic transformation. We found that neither expression of the mutant C-terminus alone nor expression of CALR lacking the C-terminus leads to cytokine-independent growth, suggesting that the novel C-terminus is necessary (but not sufficient) for transformation. We therefore generated an extensive series of truncation, domain deletion and point mutations within the C-terminus and assessed their respective transforming capabilities. Surprisingly, we found that the oncogenic activity of mutant CALR is not encoded within a specific sequence or domain of the mutant C-terminus. Rather, we found that the positive electrostatic charge of the mutant C-terminus is critical for its transforming capacity. Mutagenizing all 18 lysine/arginine residues (positively charged) within the C-terminus to a neutral glycine residue abrogates CALR-del52 transformation activity. In contrast, mutagenizing the 18 non-lysine/arginine residues within the C-terminus to glycine does not affect transforming activity, a remarkable finding considering that, in this mutant, 50% of the amino acids have been modified. Finally, using co-immunoprecipitation assays we found that mutant CALR, but not wild-type CALR, physically interacts with MPL, and that neither the mutant C-terminus alone nor mutant CALR lacking the C-terminus can bind to MPL. This suggests that the tertiary structure of mutant CALR is required for binding to MPL. Moreover, we found that the ability of our engineered CALR mutants to bind MPL perfectly correlates with their ability to mediate transformation, suggesting that the interaction with MPL is critical for mutant CALR-mediated transformation. Together, our findings elucidate a novel mechanism of pathogenesis in MPN and provide insights into how CALR mutations drive the development of MPN. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2413 Somatic mutations in IDH1 and IDH2 are common in normal karyotype AML as well as in other clonal myeloid disorders and several solid tumors. Mutant IDH overproduces the R-enantiomer of 2-hydroxyglutarate, (R)-2HG (Dang, et al Nature 462: 739, 2009), which is hypothesized to alter the epigenetic landscape of cancer cells by inhibiting the activity of α-ketoglutarate-dependent enzymes, including the TET family of 5-methylcytosine hydroxylases and the jumonji-domain-containing family of histone demethylases. There is considerable interest in developing inhibitors of mutant IDH in the hope that, by decreasing (R)-2HG production in cancer cells, their epigenetic regulation can be restored. However, there is, to date, no evidence that transformation by mutant IDH is reversible or that inhibiting production of (R)-2HG has any effect on cancers harboring IDH mutations. Herein we report that in two different myeloid transformation assays, transformation by (R)-2HG and mutant IDH1 is reversible by removal of (R)-2HG. We previously reported that stable expression of a tumor-derived mutant IDH1 (IDH1R132H) induces growth factor independence and blocks EPO-induced differentiation in the human TF-1 erythroleukemia cell line, and that treatment of TF-1 cells with a cell-permeable form of (R)-2HG, TFMB-(R)-2HG, is sufficient to recapitulate this phenotype (Late Breaking Abstract #LBA-4, ASH 2011). We have extended these studies and found that transformation by TFMB-(R)-2HG is reversible and that this reversibility is influenced by the duration and intensity (dose) of (R)-2HG exposure. We developed a second myeloid transformation assay using a murine myeloid leukemia cell line that is transformed by expression of a HoxB8-ER fusion protein when cultured in the presence of estrogen. Upon estrogen withdrawal, the cells undergo monocytic differentiation and apoptosis. We expressed wild-type IDH1 or IDH1R132H in the cells and found that cells expressing wild-type IDH1 differentiate normally, but cells expressing IDH1R132H do not upregulate monocytic markers CD11b/Mac1 and Gr1 upon estrogen withdrawal. Furthermore, treatment of the IDH1R132H-expressing cells with an inhibitor of mutant IDH1 restores their ability to undergo monocytic differentiation upon estrogen withdrawal. Our findings suggest that continued exposure to (R)-2HG is required to maintain the cellular changes induced by mutant IDH, and further suggest that targeting (R)-2HG production may have therapeutic efficacy in the treatment of cancers harboring IDH mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract LBA-4 Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities. In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. Disclosures: Kaelin: Fibrogen: Consultancy, Equity Ownership.

Description: Background: Purpura fulminans (PF) is a rare but devastating complication of sepsis in which patients experience overwhelming systemic thrombosis leading to end organ damage, distal limb ischemia, and death. Survivors are often left with lifelong disfigurement and/or disability due to severe acral tissue injury and the need for amputation of necrotic extremities. Given that most patients who develop PF have no known underlying medical conditions and are much younger than those typically afflicted with sepsis, we sought to investigate whether genetic determinants predispose individuals to developing PF. Methods: We performed whole exome sequencing on the Boston PF Cohort (N=40). We used pathway-based collapsing analysis (PBCA) combined with mutational burden testing to evaluate for enrichment of rare coding variants in the complement system using as a comparator unselected patients with sepsis from the NHLBI ARDSnet iSPAAR cohort (N=87). Several novel variants in integrin complement receptors were identified and subsequently cloned and functionally characterized. Results: Patients in the Boston PF Cohort were relatively young at the time of presentation, with a median (IQR) age of 37.5 (20.3-58.8) years. Patients were afflicted with gram negative organisms 45% of the time, while 27.5% were infected with gram positive organisms. In another 27.5%, no pathogen was identified. PF patients had strikingly abnormal markers of severe sepsis and coagulopathy, including the following median (IQR) values: lactate 7.1 (5.3-11.8) mmol/L, platelet count 28,000 (15,000-48,500) per μl, aPTT 68.0 (48.3-142.5) seconds, INR 2.8 (1.9-4.0), and protein C activity 26.0% (13.5-38.5). No patient had a known congenital immune defect. Whole exome sequencing (mean read depth 80X) identified 30 unique heterozygous complement system variants in 26/40 (65%) patients with PF (Figure 1A-B). Variants in this pathway were highly enriched in PF patients compared to the control cohort (P