ALBERT

Description: Mixed Lineage Leukemia gene rearrangements (MLL-r) account for nearly 10% of human acute leukemia cases and are generally associated with poor prognosis. Previous studies have revealed an essential role of the histone H3K79 methyltransferase Disruptor of Telomeric Silencing-1 Like (DOT1L) in MLL-r leukemogenesis. Our recent report (Chen et al. 2015 Nature Medicine) further identified a role for histone acetylation in DOT1L dependent gene expression driven by MLL-fusion proteins including MEIS1 and HOXA cluster genes. A first-in-human Phase I clinical trial demonstrated clinical activity of DOT1L inhibition in MLL-r leukemia patients, thus providing a potential opportunity for treating these malignant diseases. Nevertheless, the incomplete silencing of the leukemic program by only targeting DOT1L motivates the need for additional and perhaps combinational approaches to improve therapies against MLL-r leukemias. To enhance the efficacy of DOT1L inhibition, we sought to identify genes whose suppression would synergize with the DOT1L inhibitors to suppress the proliferation of mouse bone marrow progenitors transformed with MLL-AF9. We conducted a pooled RNAi screen using a customized library composed of 2,252 shRNA targeting 468 epigenetic regulators (i.e. writers, readers, and erasers of chromatin modifications; Fig 1). The integrated shRNA sequences were assessed using high-throughput sequencing. By comparing the change in frequency of each shRNA construct cultured in control vs. an IC50 DOT1L inhibitor EPZ4777, we identified several candidate modulators of DOT1L dependency, which had multiple shRNAs selectivity depleted only in the DOT1L suppressed condition. Notably, using a network correlation study, we found that one of the top candidate genes Plant Homeodomain Finger Protein 20 (PHF20) is highly associated with histone acetylation in the mammalian epigenome. Knockdown of PHF20 drastically increased the sensitivity of MLL-AF9 leukemic blasts to DOT1L inhibitors through enhanced myeloid differentiation and reduced cell proliferation, colony formation, and re-plating capacity. Similar phenotypes were also observed in PHF20-deficient MLL-AF9 cells generated by CRISPR/Cas9-mediated gene knockout. PHF20 is an epigenetic adaptor protein that has no predicted enzymatic activity. To investigate the role of PHF20, we conducted a CRISPR functional domain screen and identified the requirement of the chromatin reader domains in PHF20, including the Tudor domains and the PHD-finger, in supporting the survival of MLL-r leukemic cells upon DOT1L inhibition. We also performed RNA-seq and found that suppression of PHF20 facilitated the silencing of the MLL-AF9 leukemic program induced by DOT1L inhibitor treatment. Chromatin immunoprecipitation and sequencing (ChIP-seq) analyses validated that PHF20 contributes to the maintenance of histone acetylation including H3K9ac and H4K16ac at MLL-AF9 target loci. In line with the profound loss of histone acetylation at MLL-AF9 target loci in PHF20-depleted cells, we found that knockdown of a known PHF20 interacting partner KAT8 (a histone acetyltransferase; also known as MOF or MYST1) phenocopies the effects observed in PHF20-knockdown cells. Finally, we showed that pharmacological inhibition of DOT1L and KAT8 synergistically suppresses the proliferation and survival of MLL-AF9 leukemic cells. These data collectively highlight the involvement of a novel DOT1L-PHF20-KAT8 axis in mammalian gene regulation and MLL-r leukemogenesis. In summary, our studies show that MLL-rearrangements may drive leukemic transformation by coordinating an epigenetic network involving several histone modifications associated with gene transcription (e.g. H3K79 methylation and H3K9/H4K16 acetylation). Our results also suggest that simultaneous targeting of multiple components of this epigenetic feed-forward loop including DOT1L and PHF20/KAT8 may provide a novel and more effective approach against MLL-r leukemia. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Description: Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.

Description: Key Points A focused RNAi screen identifies Dhx9 as a regulator of ABT-737 sensitivity in Eµ-myc/Bcl-2 lymphomas. Dhx9 suppression activates an apoptotic signal through the Chk1/p53 replicative stress pathway in Myc-driven cells.

Description: Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations. Disclosures No relevant conflicts of interest to declare.

Description: Key Points In a Ph+ ALL mouse model, dasatinib inhibition of the BCR-ABL kinase resensitizes residual leukemic B cells to Janus kinase inhibition. Dasatinib, ruxolitinib, and dexamethasone together limit emergence of dasatinib-resistant BCR-ABL mutants and extend long-term survival.

Description: Chemoresistance remains a major barrier to successful clinical treatment of acute myeloid leukemia (AML). However, the underlying molecular mechanisms of chemoresistance in AML are largely unknown. Previously, we reported that loss of tumor suppressor genes, Nf1 and Trp53, can confer upon myeloid leukemia cells partial resistance to cytarabine (Ara-C), a mainstay of AML chemotherapy. Microarray gene expression profiling revealed that a group of lysosome function-related genes, and a major regulator of apoptosis, Bcl-xL, were upregulated in in vitro Ara-C resistant myeloid leukemia cell lines. We now report that the lysosome number and size is increased in resistant cells. Also, overexpression of Bcl-xL in both murine and human myeloid leukemia cell lines increased Ara-C resistance to a variant extent in different lines. Furthermore, we found that the key enzyme involved in intracellular activation of Ara-C, Cda, can do so, too, but to a less extent than Bcl-xL. Interestingly, in our Ara-C resistant AML cell lines which were retrovirally induced in BXH-2 stain of mice, we were able to detected new proviral insertion sites which may cause mutations associated with the leukemia resistance feature. We have now examined the possibility of modeling AML chemoresistance in mouse, and through this model, to identify and characterize AML relapse-related genetic abnormalities. During establishing such a model, treatment dosage, intervals, courses, routes of administration, the number of injection, food preparation and supplements have been considered for testing. Ara-C treatment caused a decrease in body weights and peripheral blood cell counts. One major lethal toxicity response, intestine deterioration, appeared to be correlated with high doses of Ara-C. We arrived at a regimen comprised of two 10-day treatment courses at 90 mg/kg/day by one intraperitoneal injection, with a 5-day break between treatment courses. This treatment course was found to give rise to 10~30% lethality due to Ara-C toxicity but it can also suppress the onset of transplanted AML disease in recipient mice for nearly two weeks compared to saline-treated mice as a control. Interestingly, the tumors collected from Ara-C treated transplants displayed higher resistance than saline treated tumors in a methylcellulose colony-forming assay. Testing of apoptosis regulatory genes for Ara-C response in primary tumors is in process.

Description: Aggressive Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease can be induced by the introduction of cultured p185 (BCR-ABL)-expressing Arf-null pre/pro-B cells into healthy, unconditioned syngeneic mice. Only 20 polyclonal donor cells are sufficient to induce lethal ALL within 30 days of their IV administration, indicating that BCR-ABL expression and Arf inactivation are sufficient to guarantee leukemogenesis in healthy recipient animals. Leukemic mice enter transient remission in response to treatment with potent second generation tyrosine kinase inhibitors (TKI) such as dasatinib (SprycelTM). However, like human patients with Ph+ ALL, the continuously treated animals ultimately relapse with the emergence of leukemic clones containing clinically relevant BCR-ABL mutations, the nature of which depends upon the intensity of TKI treatment. Premature withdrawal of dasatinib when animals are in remission results in re-emergence of leukemia; surprisingly, leukemic B cells recovered from these animals lack BCR-ABL mutations and remain sensitive to dasatinib ex vivo. Hence, minimal residual disease depends upon salutary signaling within the hematopoietic microenvironment. In agreement, the response to TKI therapy can be significantly improved by abrogating cytokine signaling through a knockdown of the common gamma chain of the cytokine receptor. Administration of the Janus kinase (JAK) inhibitor ruxolitinib (Jakafi™) mimics this response. Although ruxolitinib demonstrated no anti-leukemic activity of its own, the overall survival of leukemic mice inoculated with 200,000 p185+ Arf-/- pre/pro-B cells was significantly extended after administration of a targeted combination therapy of ruxolitinib and dasatinib in comparison with mice treated with dasatinib alone. Addition of dexamethasone further reduced the leukemic burden, prevented CNS relapse, and led to prolonged survival. This implicates prevention of minimal residual disease and relapse by a non-toxic combination of targeted treatments. These studies have provided a rationale for a Phase I/II clinical trial employing these agents, particularly in older patients who are ineligible for bone marrow transplantation or do not tolerate cytotoxic chemotherapy. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

Description: Introduction Premature senescence is a permanent proliferative arrest that occurs in response to oncogenic signaling or DNA-damaging chemotherapy. Although tumor cell senescence has been recognized as a prognostically relevant contribution to long-term outcome post-therapy in hematological tumor models, therapeutic utilization of senescence is still hampered by an incomplete understanding of biological properties and long-term fate of senescent tumor cells in patients. Interestingly, senescence-regulating factors have recently been shown to limit reprogramming of somatic cells to pluripotency, and to protect stem cell compartments from premature exhaustion. Hence, we explore here whether cellular senescence and stemness may functionally overlap, thereby potentially equipping arrested cells with latent self-renewing potential. Methods Stem cell-related features (stem cell gene signatures, Sca-1 expression, ALDH and ABC transporter activity) were analyzed in primary apoptosis-blocked Eµ-myc transgenic B-cell lymphomas, which enter treatment-induced senescence (TIS) in response to standard antineoplastic agents. Several (e.g. Suv39h1- or p53-based) genetic models were established, in which TIS occurred in a conditional and reversible fashion. Clonogenicity, proliferative and repopulating assays were performed in vitro and in vivo, comparing individual lymphomas that grew out of senescence (“Previously Senescent”, PS) with matched lymphoma cells that equally received chemotherapy, but were incapable of entering TIS (“Never Senescent”, NS). In a mouse model of T-cell acute lymphoblastic leukemia (T-ALL), stem cell markers and tumor initiating potential were assessed in a flow-sorted non-self-renewing leukemia cell population after senescence induction by chemotherapy. Human hematological cancer cell lines and tumor samples obtained from B-cell lymphoma and acute myeloid leukemia (AML) patients were analyzed for stem cell features after exposure to senescence-inducing chemotherapy in vitro. Results Senescent mouse lymphomas were strongly skewed towards an increased expression of an adult tissue stem cell signature, distinct stem cell markers and functional stemness properties. Upon release from conditional senescence, PS cells resumed proliferation and rapidly exceeded the proliferative, clonogenic and tumor-initiating capacity of NS cells. Interrogation of self-renewal-relevant cascades revealed activation of and dependence on canonical Wnt signaling in senescence, as blocking of this pathway reduced the growth of PS, but not NS cells. Moreover, TIS-related stemness occurred independent of secretable factors. Strikingly, in a murine T-ALL model, temporary senescence enforcement re-programmed non-stem leukemia cells into leukemia stem cells, allowing PS bulk leukemia cells to de novo initiate leukemias in recipient mice. These results were supported by consistent findings in human hematological cancer cell lines as well as primary human B-cell lymphoma and acute myeloid leukemia samples. Conclusions Our findings uncover senescence-associated stemness as a detrimental capability which is latently enriched for by chemotherapy in lymphoma and leukemia. The aggressive growth potential might become evident when senescent cells occasionally acquire alterations that allow them to re-enter the cell-cycle, thereby unleashing the tumor-promoting potential of a biological program so far considered to operate as a tumor-suppressive mechanism. However, targeted intervention at stemness-related signaling cascades in senescence may open novel therapeutic options for apoptosis-resistant lymphoma and leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. Disclosures No relevant conflicts of interest to declare.

Description: Mutations in genes encoding RNA splicing factors constitute the most common class of genetic alterations in patients with myelodysplastic syndromes (MDS). These occur as heterozygous point mutations at specific amino acid residues in SF3B1, SRSF2, and U2AF1, and are almost always mutually exclusive with one another. Recent studies have identified that mutations in each of these genes results in activation of the innate immune signaling through altered splicing of mRNAs encoding key enzymes in this pathway. Now, through an unbiased genetic screen as well as focused genetic studies, we have identified that SF3B1-mutant MDS depends on aberrant immune signaling for cell survival. Recent work has identified that aberrant splicing of MAP3K7 (also known as TAK1; TGF-b Activating Kinase 1) is pervasive across SF3B1-mutant human and mouse cells and results in reduced MAP3K7 protein expression and increased NF-κB signaling. Consistent with this, Map3k7 haploinsufficiency in myeloid cells is known to cause myeloproliferation, while at the same time, complete loss of Map3k7 is intolerable for hematopoietic cells. We therefore hypothesized that partial inhibition of MAP3K7 might preferentially impact SF3B1-mutant cells. To test this hypothesis, we generated mice with inducible deletion of 1 or 2 copies of Map3k7 (Mx1-cre Map3k7fl/+,Mx1-cre Map3k7fl/fl) alone or in the presence of mutant Sf3b1K700E (Mx1-cre Map3k7fl/+Sf3b1K700E/+,Mx1-cre Map3k7fl/flSf3b1K700E/+), along with all controls (Mx1-cre Sf3b1+/+ Map3k7+/+ (Wildtype; WT) and Mx1-cre Sf3b1K700E/+ mice). We then performed bone marrow transplantation (BMT) to assess the effect of Map3k7 deletion on aberrant hematopoiesis driven by mutant SF3B1. Consistent with prior reports, heterozygous deletion of Map3k7did not affect repopulating potential in BMT assays compared to controls while homozygous deletion of Map3k7 resulted in complete failure of hematopoiesis (Figure A). Interestingly, however, in the presence of Sf3b1K700E mutation, deletion of a single copy of Map3k7 completely rescued the hematopoietic defects characteristic of mutant SF3B1 in both mature and immature cells (Figure B-C). These data suggest that inhibition of residual MAP3K7 function may preferentially target SF3B1-mutant MDS cells. In parallel to the above studies, we also performed a negative selection RNAi screen to uncover novel genetic dependencies in SF3B1-mutant myeloid neoplasms. We performed pooled lentiviral infection of shRNAs targeting ~2,200 genes encoding proteins which are drug targets ("The Druggable Genome") under the control of a doxycycline-inducible vector in isogenic K562 cells expressing the two most commonly occurring SF3B1 mutations, SF3B1K666N and SF3B1K700E, from the endogenous SF3B1 locus. Two individual clones per SF3B1-mutant line were used to improve the robustness of the screen. On Day 21 following shRNA activation, genes with ≥3 shRNAs depleted in SF3B1-mutant cells while remaining unchanged in parental K562 cells were selected. This identified 101 candidates that are potentially synthetic-lethal with SF3B1 mutation (Figure D). Interestingly, pathway analysis of these potential candidates revealed of genes involved in immune and inflammatory signaling as well as in metabolic processes (Figure E). Further target validation was performed using in vitro competitive growth assay in K562 cells, and another set of SF3B1 isogenic lymphoid leukemia cell lines (NALM-6) expressing the same mutations. This revealed consistent dependency of SF3B1-mutant cells on STAT1, an essential component of the interferon (IFN) signaling pathway (Figure F). Upon exposure to Type-I IFNs, SF3B1-mutant K562 cells showed increased transcriptional response in IFN-responsive genes containing interferon stimulated response elements (ISREs) compared with SF3B1 WT cells (Figure G). These data highlight that SF3B1-mutant cells are hyper-responsive to IFN signaling and require intact IFN-signaling responses for cell survival. Taken together, the above studies indicate that sustained IFN signaling as well as activated innate immune signaling downstream of TAK1 are required for the survival of SF3B1-mutant myeloid cells. These results therefore have important therapeutic implications as they suggest that pharmacologic inhibition of STAT1/Type I IFN activation and/or TAK1 may serve as important therapeutic agents for SF3B1-mutant MDS. Figure. Figure. Disclosures No relevant conflicts of interest to declare.