MLL-AF9 Leukemogenesis Research Articles

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

JMJD1C Interacts with the Ncor Corepressor Complex and Is Required for Human MLL-AF9 Leukemogenesis

MLL rearranged leukemia (MLLr) is a subtype of Acute Myeloid Leukemia (AML) in which the N-terminus of the Mixed Lineage Leukemia (MLL1) gene fuses with one of over 60 fusion partner genes. The most common fusion in AML is MLL-AF9. MLLr leukemia has an intermediate to poor prognosis. Previous work has identified JMJD1C, a jumonji containing lysine demethylase, as a target of MLL-AF9 fusion oncoprotein, and studies in mice have shown JMJD1C is required for MLL-AF9 and HOXA9/MEIS1 leukemogenesis. Further, our lab utilized CRISPR(clustered regularly interspaced short palindromic repeats))/Cas9(CRISPR-associated protein-9 nuclease) to show the Jumonji (JmjD) and Zinc finger domains (ZFD) of JMJD1C are important for its function in mouse MLL-AF9 leukemia. While the role of JMJD1C in mouse MLL-AF9 leukemia is well documented, its role in human disease is incompletely understood. We studied the role of JMJD1C in human AML using human CD34+ cord blood derived MLL-AF9 (CD34+ MA9) model. We transfected CD34+ MA9 cells with a combination of three sgRNAs targeting genomic regions of JMJD1C, or sgRNA against Renilla as control to knockout JMJD1C and assessed the effect on leukemia cells in vitro. We observed significant increases in apoptosis and levels of mature markers CD13 and CD14 in JMJD1C knockout versus control cells by flow cytometry. Experiments are ongoing to assess knockout of JMJD1C on CD34+ MA9 leukemogenesis in vivo. To study the specific requirement of JmjD and ZFD domains in human leukemia, we transduced CD34+ MA9 leukemia cells with single sgRNAs targeting the JmjD or ZFD. We performed competitive transplants and found significant depletion of cells harboring JmjD and ZFD sgRNAs compared to controls, suggesting they are functionally important for human MLL-AF9 leukemogenesis in vivo. Finally, we performed RNA-seq on CD34+ MA9 cells transduced with sgRNA against JmjD or ZFD domain, which showed positive enrichment of apoptotic pathways and significant negative enrichment for human MLLr leukemia, AML, and Hematopoietic Stem Cell pathways in ZFD and JmjD mutated cells. To further understand the mechanism behind the requirement of JMJD1C in human AML, we sought to identify novel interacting proteins of JMJD1C by using BioID, an unbiased proximity ligation approach. Thirteen unique proteins were identified by Mass Spectrometry as JMJD1C interacting proteins including three members of the nuclear co-repressor complex: NCOR1, TBL1X, and TBL1XR1. NCOR1 has been shown to be required for MLL-AF9 leukemogenesis and, together with NCOR complex member HDAC3, binds promoters of myeloid genes to regulate myeloid cell differentiation during leukemogenesis. A phage display assay of JMJD1C was also conducted and confirmed an interaction between JMJD1C and NCOR1. We further examined the interaction in MLLr leukemia cell lines, MOLM13 and THP1, as well as, non MLLr AML cell lines, NB4 and Kasumi-1, which harbor oncogenic fusion PML-RARa and AML-ETO respectively to determine if the interaction is specific to MLLr or applies to other AML types. JMJD1C and components of the NCOR complex reciprocally co-immunoprecipitated in all four cell lines. Moreover, JMJD1C co-fractionated with the NCOR complex in size exclusion chromatography in all lines. Taken together, these results prove an interaction between JMJD1C and NCOR1 that is found in diverse AML cell lines. <Overall, this study demonstrates JMJD1C is required for human AML MLL-AF9 leukemogenesis and uncovers an interaction between JMJD1C and the NCOR complex in human AML cells. Currently, we are studying the functional importance of this interaction.

Differential roles of BAF and PBAF subunits, Arid1b and Arid2, in MLL-AF9 leukemogenesis.

The Switch/Sugar Non-Fermenting (SWI/SNF) nucleosome remodeling complexes play important roles in normal development and in the development of various cancers. Core subunits of the SWI/SNF complexes have been shown to have oncogenic roles in acute myeloid leukemia. However, the roles of the unique targeting subunits, including that of Arid2 and Arid1b, in AML leukemogenesis are not well understood. Here, we used conditional knockout mouse models to elucidate their role in MLL-AF9 leukemogenesis. We uncovered that Arid2 has dual roles; enhancing leukemogenesis when deleted during leukemia initiation and yet is required during leukemia maintenance. Whereas, deleting Arid1b in either phase promotes leukemogenesis. Our integrated analyses of transcriptomics and genomic binding data showed that, globally, Arid2 and Arid1b regulate largely distinct sets of genes at different disease stages, respectively, and in comparison, to each other. Amongst the most highly dysregulated transcription factors upon their loss, Arid2 and Arid1b converged on the regulation of Etv4/Etv5, albeit in an opposing manner while also regulating distinct TFs including Gata2,Tcf4, Six4, Irf4 and Hmgn3. Our data demonstrate the differential roles of SWI/SNF subunits in AML leukemogenesis and emphasize that cellular context and disease stage are key in determining their functions during this process.

Both Arid1b and Arid2 Are Tumor Suppressors in MLL-AF9 Leukemogenesis

ATP-dependent chromatin remodeling complexes, including BAF (BRG1 associated factor) and PBAF (Polybromo-associated BRG1 associated factor), play major roles in development and disease. These complexes contain multiple subunits and utilize ATP to remodel nucleosomes and, as a result, affect downstream processes. Subunits of the BAF and PBAF complexes are frequently mutated in various cancers, including in acute myeloid leukemia. However, the role of distinct members of these complexes in leukemogenesis is poorly understood. BAF and PBAF complexes are distinguished from each other by unique AT-rich interacting domain (ARID) subunits, ARID1A/B and ARID2, respectively. Recently, Arid2 was suggested to have tumor suppressive functions in MLL-AF9 leukemogenesis, however the role of Arid1b is unknown. In this study, we further evaluated the tumor suppressor roles of Arid2 and Arid1b in MLL-AF9 leukemogenesis. To study the effect of the loss of Arid2 and Arid1b on MLL-AF9 leukemia in vitro, hematopoietic stem and progenitor cells from Arid1bfl/fl, Arid2fl/fl and wild type (control) mice were transformed by retroviral expression of MLL-AF9. Arid1b or Arid2 was then deleted by retroviral delivery of Cre-recombinase and cellular phenotypes were characterized by flow cytometry. In vitro characterization revealed Arid1b-/-and Arid2-/-MLL-AF9 cells have similar growth rates to control cells and no changes in cell cycle status and apoptosis. To study leukemogenesis in vivo, two hematopoietic specific conditional knockout mouse models, Vav1Cre, a constitutively active Cre-recombinase, and Mx1Cre, an inducible Cre-recombinase, were used. Primary and secondary transplant recipient mice were assessed for survival, disease burden, and leukemia stem cell frequency. In primary transplantation, leukemic initiation was not altered by the loss of the Arid2. Overall survival of primary recipients and disease burden in moribund mice (percentage of GFP positive MLL-AF9 cells in bone marrow/spleen and spleen weight) were similar to control counterparts. However, in limiting dilution secondary transplantation of Arid2fl/flVav1Cre (Arid2-/-) and wild type Vav1Cre (control) MLL-AF9, median survival of the Arid2-/- cohort decreased 1 to 2 weeks in comparison to the control cohort and there was a two-fold increase in leukemia initiating cell frequency upon deletion of Arid2. Together these results support the previous indication that Arid2 is a tumor suppressor in MLL-AF9 leukemic initiation. To gain insight into the function of Arid1b in MLL-AF9 leukemogenesis, we performed primary transplantation of Arid1bfl/flcells transduced with MLL-AF9. Our data showed that loss of Arid1b enhanced leukemic initiation in the MLL-AF9 leukemia model. One-month post induction with pIpC, Arid1bfl/fl Mx1Cre MLL-AF9 cells accumulated faster in the peripheral blood and primary recipients exhibited decreased median survival compared with wild type Mx1Cre controls. Percentage of GFP+ leukemia cells was higher in the spleen of moribund mice in the Arid1bfl/fl Mx1Cre cohort, compared with the control cohort. Taken together, our data suggests Arid1b, in addition to Arid2, acts as a potential novel tumor suppressor in MLL-AF9 leukemogenesis. To study the mechanism of Arid1b and Arid2's function in MLL-AF9 leukemogenesis, RNA-sequencing of leukemic bone marrow cells was conducted. Consistent with the previous study, Arid2 null leukemia cells had increased Gata2 expression and decreased p57 expression. Gene Set Enrichment Analysis of Arid1b null cells showed enrichment of oncogenic signatures including ATM, Cyclin D and macrophage development as potentially contributing to the underlying tumor suppressive mechanism of Arid1b. In summary, our study uncovered Arid1b, in addition to Arid2, as a potential novel tumor suppressor in MLL-AF9 leukemia. Our study, together with published studies on the requirement of other subunits (SMARCA4, SMARCD2 and DPF2) of the BAF and PBAF complexes in MLL-AF9 leukemia, reveal opposing roles of the components of the BAF and PBAF complexes in leukemogenesis, the mechanism of which require further investigation. Disclosures No relevant conflicts of interest to declare.

Critical Role of Jumonji Domain of JMJD1C in AML Leukemogenesis

MLL-rearranged leukemias are found in 5-10% of adult leukemias and over 70% of infant leukemias and are associated with intermediate to poor prognosis. We have recently shown that JMJD1C, a Jumonji domain containing lysine demethylase (KDM), is important for leukemia stem cell (LSC) function in MLL-AF9 and HOXA9 leukemias but dispensable for normal hematopoietic stem cell function, therefore a potential therapeutic target for acute myeloid leukemia (AML).To determine which domains within JMJD1C are essential for its function in AML, we performed a CRISPR/Cas9 negative selection screen against its coding sequence using guide RNAs (gRNA) tiled at ~ 30bp intervals. Specifically, gRNAs were cloned into a lenti-viral vector with a TdTomato marker gene, transduced into a clonal Cas9 expressing mouse MLL-AF9 leukemia and percentage of gRNA positive cells was analyzed by flow cytometry in a time course. JMJD1C Jumonji and Zinc finger domain (ZFD) and surrounding regions exhibited the most depletion at day 14 compare to day 2, comparable or higher than that of the positive control. To investigate the effect of mutating Jumonji and ZFD on MLL-AF9 leukemogenesis, we subject gRNA transduced MLL-AF9 Cas9 cells to colony forming cell assay. Mutating Jumonji or ZFD impaired colonegenic potential of MLL-AF9 Cas9 cells as evident by a significant reduction in the number of colonies. The resulting colonies appeared smaller and diffused, indicative of cell differentiation. Consistently, we observed impaired proliferation, increased apoptosis and increased expression of the myeloid differentiation marker Mac1 upon mutating these domains. Moreover, mutating Jumonji and ZFD prolonged survival of recipient mice compare to control in transplantation experiment. Finally, we performed chromatin-immunoprecipitation followed by sequencing (ChIP-seq) on H3K27, K36 and K9 methylation in JMJD1C knockout versus floxed MLL-AF9 leukemia cells to identify histone marks that correlate with JMJD1C activity. We observed increased H3K36 trimethylation (me3) level on JMJD1C regulated genes loci in the knockout cells compare to controls. Similar increase was observed upon mutating Jumonji and ZFD using CRISPR/Cas9 in ChIP assay. Using Gene Set Enrichment Analysis (GSEA), we found enrichment of JMJD1C regulated genes in the knockout cells in H3K36me3 ChIP-seq data but not H3K27me3 or H3K9 methylation data. Together, these data suggest that Jumonji and ZFD of JMJD1C are most critical for its function in AML and that methylated H3K36 can be used as a marker for JMJD1C activity at gene loci.Next, we performed single cell RNA-seq to examine the transcriptome change upon mutating the catalytic Jumonji domain. Our analysis showed a hierarchy within control leukemia cells with one cluster bearing more LSC property than the other. Upon mutating Jumonji domain, leukemia cells are driven into differentiated states that bear either more granulocyte or monocyte gene signatures. Using MAST, a single cell transcriptome GSEA tool, we observed enrichment of KRAS signatures in Jumonji mutated cells. This observation in addition to the increased expression of the IL-3 receptor subunit genes Csf2ra/b led us to examine whether the IL-3 signaling pathway is affected in these cells. Western blotting of IL-3 down signaling molecules ERK1/2 and STAT5 showed elevated phosphorylation in Jumonji mutated cells compare to controls both at basal level and in response to IL-3 stimulation. Consistently with this finding, Jumonji mutated cells showed increased sensitivity to inhibitors against RAS (Tipifarnib), MEK (Selumetinib) and JAK (Tofacitinib and Ruxolitinib). Finally, by mining published shRNA and gRNA screen, we observed that mouse as well as human MLLr leukemia cell lines with activating RAS mutations showed decreased or diminished response to knockdown or mutation of JMJD1C suggesting that the presence of RAS mutations render them resistant to JMJD1C modulation.Overall, we show that enzymatic domain of JMJD1C, the Jumonji domain, mediates its function in AML leukemogenesis, therefore can be targeted for potential therapeutic intervention. In addition, we uncovered a novel interplay between JMJD1C and IL-3 signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells

AbstractThe concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)–enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9–mediated leukemic disease in mice.

Abstract 2694: Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis

Abstract Chromosomal rearrangements of the Mixed-Lineage Leukemia (MLL) gene are found in 5-10% of all patients with acute leukemia and associated with a poor prognosis. MLL-rearrangements are more frequently present in pediatric and infant patients where AF9 is one of the most common fusion partners. In order to identify novel druggable targets in MLL-AF9 rearranged leukemia, we conducted a chromatin regulator focused RNAi screen in murine MLL-AF9 leukemia cells and found hairpins targeting (K)Lysine Acetyltransferase 8 (Kat8, also known as Mof) and the previously identified target Bromodomain Containing 4 (Brd4), to be the most potent suppressors of cell growth. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and member of the MYST family of histone acetyltransferases (HATs). MOF has been shown to be crucial for murine embryogenesis and is a cell-type dependent regulator of chromatin state and various cellular processes such as T-cell differentiation, DNA damage response and cell cycle progression. Using a conditional murine Mof knockout system, we studied the role of MOF in MLL-AF9 leukemogenesis in detail. In vitro inactivation of Mof in MLL-AF9 transformed mouse hematopoietic stem and progenitor cells led to impaired colony-forming capacity. The specificity of this phenotype was shown by expression of exogenous full-length Mof, which fully rescued transformed cells from the dramatic phenotype. Inactivation of Mof in vivo, lead to reduced tumor burden and prolonged survival of mice bearing MLL-AF9 leukemia cells. RNA sequencing data comparing MLL-AF9 cells with homozygous Mof loss to a wild type control, showed a significant enrichment of genes within the apoptosis (NES 1.98, FDR-q &amp;lt;0.0001) and p53 (NES 2.23, FDR-q &amp;lt;0.0001) pathway. These gene expression data suggest that the importance of MOF in MLL-AF9 leukemogenesis may be through interaction with p53, inducing proliferation and suppressing apoptosis. In addition, we found a reduction of actively cycling cells and a loss of global H4K16 acetylation (H4K16ac) upon Mof knockout. In line with this finding of H4K16ac loss, rescue experiments with HAT domain mutated MOF illustrated that the HAT activity of MOF is indispensable for MLL-AF9 leukemia maintenance. Finally, experiments with the selective MYST protein HAT inhibitor MG149, showed a strong anti-proliferative effect on murine, as well as human MLL-AF9 leukemia cell lines, and MG149 inhibition induced global H4K16ac loss in these cells. These results indicate that MOF HAT activity is required for MLL-AF9 leukemia maintenance. Our data further suggest that MOF HAT activity may be a good target for new small molecule inhibitor development for the treatment of patients with MLL-AF9 rearranged leukemia. Citation Format: Daria G. Valerio, Haiming Xu, Chun-Wei Chen, Takayuki Hoshii, Meghan Eisold, Christopher Delaney, Monica Cusan, Aniruddha J. Deshpande, Chun-Hao Huang, Amaia Lujambio, George Zheng, Tej K. Pandita, Scott W. Lowe, Scott A. Armstrong. Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2694.

Balancing Proliferation, Differentiation, and Survival: Powerful Genetic and RNAi Technologies Reveal Essential microRNA Signaling for Leukemic Progenitor Cell Fitness

Acute myeloid leukemias (AML) are a heterogeneous group of malignancies with distinguishing gene and microRNA (miRNA or miR) expression profiles. In particular, expression of the miR-196 family of miRNA is significantly associated with a large fraction of AML expressing HOX gene signatures (e.g. NPM1c mutant, 11p15 and 11q23 cytogenetic abnormalities) and is prognostically instructive. However, the requirement for miR-196 in hematopoietic cell immortalization, malignant transformation, and leukemogenesis is not understood. We note that miR-196a-1 and miR-196b are both induced upon MLL-AF9 expression, and that miR-196b is a direct MLL-AF9 target gene. To genetically evaluate the necessity of miR-196 for MLL-AF9 tumorigenesis, we varied the number of miR196-encoding alleles and tested the capacity for marrow transformation by MLL-AF9. Specifically, we transduced bone marrow cells from wild-type (WT), miR-196b+/-, and miR-196a-1-/- b-/- double-knockout (DKO) mice with retroviruses expressing MLL-AF9 to limit (miR-196b+/-) or completely eliminate (DKO) miR-196 activity. All groups were immortalized in vitro, as evidenced by the formation of morphologically blast-like colonies, accompanied by serial replating in methylcellulose colony assays. Moreover, we found similar deregulation of HoxA9 and Meis1 expression. Since these are two essential MLL-AF9 target genes, we conclude that the MLL-oncoprotein complex must be functional without miR-196. However in vivo, despite similar levels of engraftment, only mice transplanted with WT or miR-196b+/- MLL-AF9 cells formed leukemia (median latency 70 and 76.5 days, respectively; mice were followed for a total of 135 days). Flow cytometric analyses of leukemic granulocyte-monocyte progenitors (GMP) harvested from miR-196b+/- MLL-AF9 moribund mice displayed a significant increase in CD11b expression as compared to WT MLL-AF9 controls. These miR-196 haploinsufficient and loss-of-function AML models genetically demonstrate that miR-196 activity is critical to fully transform and block differentiation of malignant progenitor cells. Next, we identified AML-relevant miR-196 targets by purifying miR-196b/RNA-target/RISC complexes in human 11q23-translocation AML cells, validating putative targets in luciferase reporter assays, then testing them in an in vivo leukemogenesisshRNA-enrichment screen. Knockdown of several miR-196b targets cooperates with MLL-AF9 to accelerate leukemogenesis, including Cdkn1b. Notably, Cdkn1b- knockdown cKit+ MLL-AF9 splenocytes from moribund mice displayed significantly decreased CD11b expression and increased colony forming potential in vitro. However, simply reducing Cdkn1b in MLL-AF9 leukemia cells did not alter the number of functional leukemia initiating cells (LIC) in an in vivo limiting-dilution analysis (suggesting that Cdkn1b- knockdown does not directly affect LIC biology). Instead, RNA-Seq analyses of Cdkn1b- knockdown MLL-AF9 leukemia cells from moribund animals showed increased expression of proliferation, cell cycle, and survival pathways with decreased expression of myeloid differentiation and apoptotic pathways. Taken together, these data suggest that during leukemogenesis miR-196 activity (through direct targets such as Cdkn1b) provides a leukemia cell fitness advantage, defined by the ability of a malignant cell to intrinsically balance the conflicting programs of proliferation/self-renewal and differentiation, resulting in survival. Given the critical role of miR-196 in MLL-AF9 transformation and leukemia maintenance, we asked whether the miR-196-Cdkn1b pathway might be a point of therapeutic intervention. Indeed, forced overexpression of Cdkn1b significantly diminished colony formation in vitro, and eliminated AML in vivo. Translating this into an RNAi therapeutic, we treated murine MLL-AF9 cells with locked nucleic acid (LNA) sequences designed to specifically block miR-196b binding to its target site in Cdkn1b mRNA. This resulted in not only significant de-repression of p27Kip1 expression, but also reduced MLL-AF9 colony formation in vitro. In sum, we have established a critical genetic requirement for miR-196b in MLL-AF9 leukemogenesis through the balanced control of growth and differentiation, identified a relevant target, and demonstrated therapeutic potential of inhibiting miR-196 binding to this single target gene. DisclosuresNo relevant conflicts of interest to declare.

JMJD1c, a Histone Demethylase, Is Required for the Maintenance of MLL-AF9 AML Leukemic Stem Cells

While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study.Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples.Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort).In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients. DisclosuresNo relevant conflicts of interest to declare.

GNA13, a Novel Component of the GPR84/Beta-Catenin Signaling Axis in AML Stem Cells, Regulates the Maintenance of MLLAF9 Leukemogenesis

Beta-catenin is required for the establishment of leukemic stem cells (LSC) in mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). Targeted inhibition of beta-catenin signaling has been hampered by the lack of pathway components amenable to pharmacological inhibition. Our recent study has identified a new beta-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. Since no GPR84-specific anti-cancer compound has been developed to date, we aim to further delineate the GPR84/beta-catenin axis in order to inhibit other components of this signaling pathway pharmacologically.Preliminary data from our lab has implicated GNA13 as a potential downstream target gene of GPR84. In this study we sought to further examine the involvement of GNA13 in the GPR84/beta-catenin signaling axis. Functional studies showed that shRNA-mediated ablation of GNA13 significantly decreased the capacity of MLLAF9-LSC to form colonies in vitro (P=0.0184). We further demonstrated that GNA13 inhibition in LSC induced a significant G1 cell cycle arrest in vitro (P=0.0192). Subsequent in vivo studies showed that GNA13 downregulation in MLLAF9-LSC significantly delayed the onset of AML (P=0.0016) and significantly decreased LSC proliferation (P=0.0066). Hence GNA13 inhibition mimics the previously described GPR84-deficient phenotype in MLLAF9-LSC (Blood 2013;122:3781). In contrast, the GPR84-deficient phenotype could be rescued in MLLAF9-LSC by GNA13 overexpression in vitro (P=0.0130) and in vivo (P=0.0012). Further analysis showed that there was a significant increase in the mitotic index of GPR84-deficient LSC transduced with GNA13 cDNA in vitro, evidenced by an increase in G2/M-phase accumulation (P=0.0030) and p-histone H3 (P=0.0040). In addition, our data demonstrated a significant increase in BrdU incorporation of GPR84-deficient LSC overexpressing GNA13 in vivo (P<0.0001).In conclusion, our studies have identified GNA13, a novel component of the GPR84/beta-catenin axis. Since this pathway is pivotal for MLLAF9 AML maintenance by sustaining LSC self-renewal, targeting the GPR84/GNA13/beta-catenin signaling axis may provide a novel and promising strategy for improving the therapy and outcome of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Structural and Functional Studies Of The Intrinsically Disordered Protein AF9 In MLL-AF9 Leukemia

Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners1. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis.We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our 15N-1H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis.1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013). Disclosures:No relevant conflicts of interest to declare.

Functional Screening Of Oncomir-196b-RISC Captured Targets Reveal Mir-Inhibition Of Tumor Suppressor Activity In MLL-AF9 Mediated Leukemogenesis

Recent observations indicate that microRNA (miR) are deregulated in cancer and are important for disease pathogenesis; however, how miR exert direct or indirect regulatory effects on intracellular signaling pathways is poorly understood. can be attributed at least in part to delayed development of technologies to capture miRNA-target RISC complexes. Target prediction algorithms use miRNA seed sequence homology scans and RNA structural information, but problems with accuracy and failure to account for critical biological factors such as cellular context diminish their utility. Acute myeloid leukemias (AML) with HoxA gene expression signatures (e.g. NPM1c mutant, 11q23 and 11p15 cytogenetic abnormalities) have distinct gene and miRNA expression profiles compared to other subgroups of AML and normal hematopoietic cells. MLL (11q23) translocations comprise approximately 10% of adult AML, and confer a poor prognosis. Thus, this subset of AML may benefit from targeted disruption of miR function through RNA therapeutic intervention. We find significantly elevated miR-196b expression in primary patient blasts, human AML cell lines, and primary murine leukemic spleenocytes harboring HoxA signatures compared to non-Hox based leukemias and normal controls. Moreover, RNA therapeutic antagonism of miR-196b function specifically kills 11q23 AML cells but not AML cells containing other chromosomal rearrangements or normal cells. To define miR-196b targets, miR-196b-mRNA RISC complexes were purified from two different cell lines then target mRNA identities were revealed by microarray analyses. We identified 422 putative leukemia-relevant miR-196b captured targets, of which 50% were predicted by algorithms. Both predicted and novel targets were independently validated by repeated miR-target capture experiments and target site sequences were confirmed by luciferase reporter assays. When miR-196b levels were modulated over a four-day time course, we quantified differential responsiveness in captured target mRNA expression patterns over time, while protein levels of the novel targets were uniformly reduced. Bioinformatic network analyses of captured targets illustrate several biochemical pathways that might be involved in leukemogenesis. To functionally delineate the leukemia-relevant miR-196b targets, we devised an shRNA screen of miR-196b captured targets in primary murine Mll-Af9 leukemia. We identified tumor suppressor Cdkn1b (p27) as a functional miR-196b target, in that knockdown of Cdkn1b mimicked miR-196b function by accelerating Mll-Af9 leukemogenesis. Importantly, RNA therapeutic inhibition of miR-196b in Mll-Af9 leukemia cells mediates p27 upregulation concomitant with robust induction of cell death. Thus, the combination of two open-ended approaches, miRNA-target-identification and functional shRNA screen validation, revealed leukemia-context specific miR-196b functions, which are not detected by standard approaches such as target mRNA modulation or prediction algorithms. Disclosures: Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

Rac GTPase Survival Signaling Through Anti-Apoptotic Bcl-2 Proteins Reveals a Target for Combination Therapy in MLL-AF9 Acute Myeloid Leukemia

Abstract 1536The Rac family of small Rho GTPases has attracted interest as a therapeutic target in hematologic malignancies due to their central role in coordinating diverse cellular processes such as adhesion, cytoskeletal organization, proliferation, and survival. Rac activity is increased in MLL-AF9 (MA9) acute myeloid leukemia, and Rac inhibition using the small-molecule NSC23766 induces apoptosis in MA9 cells. We recently found that loss of Rac2 delays development of MA9 leukemia in a murine genetic model. Furthermore, latency of disease can be rescued by ectopic expression of Bcl-xL in the Rac2 knockout cells implicating Rac2 regulation of pro-survival Bcl-2 proteins in MA9 leukemogenesis. Whether Rac survival signaling through Bcl-2 proteins can be exploited therapeutically in the developed MA9 leukemia is untested. We hypothesized that Rac2 signaling is critical for MA9 leukemia cell survival, and that inhibition of Rac or downstream Bcl-2 proteins could be an effective therapeutic strategy alone or in combination. We tested our hypothesis in MA9 cells derived from human CD34+ umbilical cord blood (UCB) cells expressing MA9 by retroviral transduction. Lentiviral knockdown was used to determine the specific contribution of Rac2 to MA9 cell survival and maintenance. Decreased levels of Bcl-xL and Bcl-2 were seen in MA9 cells expressing Rac2-targeting shRNA, compared to non-targeting control. Rac2 knockdown induced apoptosis and impaired growth of MA9 cells in culture. Furthermore, Rac2 deficiency reduced in vitro colony-forming ability indicating impairment of the clonogenic MA9 cell. MA9 cells expressing two different Rac2 shRNA vs. non-targeting control were injected into NOD/LtSz-scid-SGM3 (NSS) mice to determine whether Rac2 deficiency impairs engraftment and progression of the MA9 leukemia stem cell in a xenotransplantation assay. Flow cytometric analysis of bone marrow aspirates showed markedly reduced MA9 engraftment in the Rac2 knockdown groups. Whereas all mice in the control group eventually died of MA9 leukemia (N=5), only one death from MA9 cells expressing Rac2 shRNA was seen in the knockdown groups (N=10). We next evaluated the effects of direct inhibition of Bcl-2 proteins downstream from Rac using the BH3-mimetic ABT-737. Three different MA9 cell lines, as well as the THP-1 cell line bearing an MLL-AF9 fusion, were highly sensitive (IC50 ∼30 nM) to ABT-737, with no toxicity seen in control UCB cells in the dose range tested. To determine in vivo efficacy, ABT-737 was administered to NSS mice engrafted with human MA9 cells. End of treatment aspirates showed a marked decrease in leukemia engraftment in the ABT-737 treatment group compared to vehicle control (<2% vs. 43%, respectively; p < 0.002). To determine whether combined inhibition of Rac and Bcl-2 proteins produces an additive effect, MA9 cell lines were treated with serial doses of ABT-737 in the presence or absence of a sub-therapeutic dose of NSC23766 (20 μM). Normal UCB cells, as well as HL-60 and two cell lines expressing the t(8;21) fusion protein AML1-ETO, were tested as controls. MA9 cell lines were exquisitely sensitive to the combined inhibition of Rac and Bcl-2 proteins, with a 2.5- to 9-fold reduction in the IC50 of ABT-737 in the presence of 20 μM NSC23766. In contrast, little to no effect was seen in any of the control cells. Our findings demonstrate for the first time that specific inhibition of Rac2 induces apoptosis and impairs the clonogenic MA9 cell in vitro and in vivo, in association with decreased expression of downstream pro-survival Bcl-2 proteins, and identify a pathway that can be exploited therapeutically. We conclude that survival signaling through Rac and Bcl-2 family proteins can be effectively targeted with small-molecule inhibitors alone and in combination in MLL-AF9 acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Rac GTPase signaling and downstream prosurvival Bcl-2 proteins as combination targeted therapy in MLL-AF9 leukemia

AbstractThe Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.

CBX8, a Polycomb Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene lead to the development of acute leukemias. Constitutive HOX gene activation by MLL fusion proteins is required for MLL-mediated leukemogenesis; however, the underlying mechanisms remain elusive. Here, we show that chromobox homolog 8 (CBX8), a Polycomb Group protein that interacts with MLL-AF9 and TIP60, is required for MLL-AF9-induced transcriptional activation and leukemogenesis. Conversely, both CBX8 ablation and specific disruption of the CBX8 interaction by point mutations in MLL-AF9 abrogate HOX gene upregulation and abolish MLL-AF9 leukemic transformation. Surprisingly, Cbx8-deficient mice are viable and display no apparent hematopoietic defects. Together, our findings demonstrate that CBX8 plays an essential role in MLL-AF9 transcriptional regulation and leukemogenesis.