ALBERT

Description: Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function.

Description: Abstract 2958 PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. Using an antibody-based protein microarray technique, we identified Citron Rho Interacting Kinase (CRIK) as a protein that was highly expressed in primary multiple myeloma (MM) cells compared to normal plasma cell. We therefore sought to investigate the functional role of CRIK in MM cells. Methods: Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM and 4 healthy control using high-throughput proteomic analysis with antibody-based protein microarray (Clontech, CA). MM.1S, OPM2, RPMI8226, and INA6 were used to perform functional validation. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform and qPCR. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, OPM2, and INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by BrdU, MTT, PI and Apo2.7 staining, and flow cytometry analysis, respectively. Results: We first showed that CRIK was overexpressed in 12 patients with MM compared to normal CD138+ cells obtained from healthy controls using high-throughput protein microarray. We further confirmed CRIK expression using immunohistochemistry in the same samples of MM patients. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Knockdown of CRIK in MM cell lines led to an anti-proliferative and pro-apoptotic effect in all MM cell lines tested. Indeed, CRIK-knockdown MM cells were characterized by a reduction of 60–80% in the proliferation rate, supported by reduction of DNA synthesis and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity of caspase-3, caspase-8, caspase-9, and parp cleavage was also demonstrated in CRIK knockdown cells compared to scramble probe transfected cells. We also showed that CRIK knockdown led to cytokinesis in MM cell lines, indicating a possible mechanism of inhibition of proliferation of these cells. Conclusion: In this study, we show that MM cells express a high level of a novel protein CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of MM cells. CRIK could be a critical therapeutic target in MM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 1943 Introduction: Plerixafor (Mozobil®), a potent CXCR4 inhibitor, is approved in combination with G-CSF to mobilize hematopoietic stem cells (HSCs) for autologous transplantation in multiple myeloma (MM) and non Hodgkin's lymphoma (NHL). Another area of investigation consists of exploring whether disruption of the CXCR4 pathway by plerixafor could potentiate the effect of chemotherapy in active disease. This study aimed to establish the maximum tolerated dose (MTD) of plerixafor in combination with bortezomib in patients who have active relapse/refractory MM. This was informed by preclinical studies showing that plerixafor induces de-adhesion of MM cells with sensitization to combination therapy with bortezomib in pre-clinical animal models. Methods: Eligibility criteria include: 1) patients with relapsed or relapsed/refractory MM with any prior lines of therapy including bortezomib, 2) measurable disease, 3) not receiving chemotherapy〉 3weeks, or biological/novel therapy for MM 〉 2 weeks. Patients with active disease received plerixafor at the recommended dose sc on days 1–6 of every cycle. Dose levels include 0.16, 0.24, 0.32, 0.40, and 0.48 mg/kg. Bortezomib was given at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. Dose levels include 1.0 and 1.3 mg/m2. Bortezomib was given 60–90 minutes after plerixafor. Patients were assessed after every cycle. Patients who had response or stable disease went on to receive a total of 8 cycles without planned maintenance therapy. 4 dose levels were initially planned at a maximum of 0.24 mg/kg plerixafor. The protocol was then modified to include 3 higher doses of plerixafor, to further evaluate the hypothesis that higher doses may induce better chemosensitization. To examine the in vivo effect of plerixafor and bortezomib on de-adhesion of MM cells and other accessory cells of bone marrow, blood samples were obtained from patients at 0, 2, 4 and 24 hours post-plerixafor injection on days 1 and 3, and time points 0, 2, and 4 hours on days 6, 10 and 13 of cycle 1 and examined for the presence of plasma cells or CD34+ cells using flow cytometry. Results: Thirteen patients have been treated to date, three in each cohort with cohort 5 currently enrolling. The median age is 60, the median lines of prior therapy is 2. All of the patients received prior bortezomib. Three patients were assessed by light chain, two patients had extramedullary disease. The median number of cycles on therapy was 5 (1-8). None of the patients came off study due to toxicity. To date, there have been no dose-limiting toxicities. Overall, the combination is very well tolerated. Grade 3 possibly related toxicities include lymphopenia (30%), hypophosphatemia (15%), anemia (8%), and hyponatremia (8%). No grade 2 or higher neuropathy has been noted in these patients. Twelve patients are evaluable for response, including 1 (8%) complete remission (CR) and 1 (8%) minimal response (MR), with an overall response rate including MR of 2 (16%) in this relapsed/refractory population. In addition, 8 (66%) patients had stable disease (SD), and 2 (18%) had progressive disease (PD). We also examined the number of plasma cells, CD34+ HSCs, and other accessory bone marrow cells (including endothelial progenitor cells and plasmacytoid dendritic cells) in the peripheral blood. Analysis of these samples is ongoing, but preliminary data indicate de-adhesion of plasma cells. Conclusions: : The combination of plerixafor and bortezomib is very well tolerated with minimal neuropathy or other toxicities. The responses observed are encouraging in this relapsed/refractory population. The ability to demonstrate transient de-adhesion of MM cells and accessory cells in vivo indicates that these cells can be separated from their protective stromal environment which may make them more sensitive to chemotherapy. This study was supported by R01CA133799-01, and by Genzyme. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees.

Description: For patients with acute myeloid leukemia (AML), failure to achieve complete remission after induction therapy or relapse after complete remission represents the major barrier to cure for both children and adults. Although genomics has revealed the mechanisms of pathogenesis of AML, the molecular mechanisms of chemotherapy resistance remain largely unknown. Using high-resolution mass spectrometry proteomics, we identified aberrant phosphorylation of MEF2C S222 in primary chemoresistant human AML specimens, and using capillary nanoimmunoassays, established its prevalence and prognostic significance in a cohort of 40 patients, spanning the major biologic subtypes of human AML, including refractory MLL-rearranged leukemias. MEF2C is a transcription factor required for hematopoietic cell fate determination. We found that expression of mutant S222A MEF2C that cannot be phosphorylated, but not phosphomimetic S222D, led to a leukemia stem cell defect in genetically-engineered MLL-AF9 mouse leukemias in vivo (Figure 1A). Similarly, loss of MEF2C phosphorylation impeded progression of human AML engrafted in immunodeficient mice in vivo. Using transcriptome and BH3 mitochondrial profiling, we found that loss of MEF2C phosphorylation induced intrinsic mitochondrial apoptosis and sensitization to both cytarabine and doxorubicin, due at least in part to the upregulation of pro-apoptotic factors BID and BMF. To determine the function of MEF2C phosphorylation in normal hematopoiesis, we used CRISPR/Cas9 genome engineering to generate transgenic mice with endogenous Mef2cS222A/S222A and S222D/S222D mutations. Both transgenic strains were born at Mendelian ratios, exhibited normal steady-state hematopoiesis, and normal reconstitution in competitive bone marrow transplants. In contrast, Mef2cS222A/S222A hematopoietic progenitor cells, transformed with MLL-AF9 oncogenes exhibited reduced colony forming capacity, as compared to transformed MEF2C wild-type litter-mate controls (Figure 1B). Thus, MEF2C S222 phosphorylation is selectively required for the survival of leukemia cells but is dispensable for normal hematopoietic function, establishing a therapeutic window for targeting MEF2C. To identify kinases that phosphorylate MEF2C and can serve as targets for therapeutic intervention, we screened recombinant serine/threonine kinases and identified the MARK kinase family as the specific enzymes that phosphorylate MEF2C S222. We found that MARK2 and MARK3 are highly overexpressed in the majority of human AML cell lines and primary patient specimens studied. We found that active MARK3, but not its inactive kinase mutants, can trans-activate MEF2C response elements using transcriptional reporter assays in a MEF2C S222-dependent manner. Importantly, MARK kinase inhibition by the ATP competitive inhibitor MRT199665, led to selective apoptosis of MEF2C-expressing human AML cell lines and primary AML patient samples, which conferred enhanced sensitivity to cytarabine (Figure 1C). Thus MEF2C phosphorylation is dispensable for normal hematopoiesis, enhances leukemia cell survival, and causes resistance to chemotherapy and susceptibility to MARK kinase inhibition. These findings establish a paradigm of rational functional therapy of AML chemoresistance. Figure 1 Figure 1. Disclosures He: Foundation Medicine, Inc: Employment, Equity Ownership. Letai:Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Gray:Gatekeeper: Equity Ownership; Petra: Consultancy, Equity Ownership; C4: Consultancy, Equity Ownership; Syros: Consultancy, Equity Ownership. Armstrong:Epizyme, Inc: Consultancy.

Description: Despite intense efforts, the cure rates of children and adults with AML remain unsatisfactory. Resistance to cytotoxic chemotherapy is the dominant cause of treatment failure. To investigate the molecular basis of primary chemoresistance, we used targeted gene sequencing using the Foundation One Heme platform to profile 405 genes in DNA and 265 genes in RNA known to be somatically mutated in hematologic malignancies in 107 primary specimens obtained at diagnosis from children and adults with cytogenetically normal AML. Comparative analysis revealed mutations of SETBP1 and ASXL1 to be enriched in patients with failure of induction chemotherapy as compared to those who achieved remission (8 out of 50 versus 1 out of 57, p = 0.01). However, the low prevalence of these alterations indicated that other genomic or functional mechanisms must mediate chemoresistance. To identify these mechanisms, we mapped kinase signaling cascades activated in chemoresistant AML cells using functional mass spectrometry proteomics. Phosphopeptides were enriched in primary leukemic blasts isolated from cytogenetically normal AML patients, with relative abundances in each patient determined from iTRAQ 8-plex reporter ions in the associated MS/MS fragment ion spectra. This analysis identified phosphorylation of the leukemogenic transcription factor MEF2C at S222 in the majority of cases. We found MEF2C to be overexpressed in cytogenetically normal AML, as well as MLL-rearranged leukemias, and to be associated with increased risk of relapse (Laszlo et al, ASH submitted). Using reporter assays, we found that phosphorylation of MEF2C S222 was both necessary and sufficient to fully transactivate MEF2C promoter elements (Fig. 1). Expression of mutant S222A MEF2C that cannot be phosphorylated, but not phosphomimetic S222D, induced mitochondrial apoptosis in human AML and genetically-engineered MLL-AF9 mouse leukemia but not in normal mouse hematopoietic progenitor cells. In syngeneic mouse transplant models, we found that MEF2C phosphorylation was required for the maintenance of MLL-AF9 leukemias in vivo. Transcriptome and proteome analysis of gene expression changes and composition of MEF2C transcriptional complexes induced by phosphorylation mutants showed that aberrant leukemia cell survival is caused at least in part by alterations in the expression of apoptotic regulators. Using a recombinant kinase screen, we identified MARK kinases as specific enzymes that phosphorylate MEF2C S222. Treatment of primary patient specimens and human AML cell lines with the MARK kinase inhibitor MRT199665 induced MEF2C downregulation and apoptosis in cells with MEF2C but not those lacking MEF2C expression (Fig. 2). Thus, aberrant MEF2C phosphorylation is associated with primary chemoresistance and induction failure, is required for enhanced leukemia cell survival, and confers susceptibility to MARK inhibition therapy. In addition, genome and proteome profiles should provide additional biomarkers and targeted therapeutic strategies to overcome chemoresistance in AML. Figure 1. MEF2C phosphorylation is required for transactivation of MEF2C promoter elements Figure 1. MEF2C phosphorylation is required for transactivation of MEF2C promoter elements Figure 2. Treatment of AML cells with MRT199665 Figure 2. Treatment of AML cells with MRT199665 Disclosures He: Foundation Medicine, Inc.: Employment, Equity Ownership. Balasubramanian:Foundation Medicine, Inc.: Employment. Zhong:Foundation Medicine, Inc.: Employment, Equity Ownership. Pavlick:Foundation Medicine, Inc.: Employment. Yilmazel:Foundation Medicine, Inc.: Employment. Stone:Merck: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Juno: Consultancy; Amgen: Consultancy; Celator: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Roche/Genetech: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Celgene: Consultancy; Novartis: Research Funding. Byrd:Acerta Pharma BV: Research Funding. Levine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy.

Description: Aberrant gene expression is a hallmark of acute leukemias. However, therapeutic strategies for its blockade are generally lacking, in large part due to the pharmacologic challenges of drugging transcription factors. MYB-driven gene trans-activation with CREB-binding protein (CBP) is required for the initiation and maintenance of a variety of acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a prototypical peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP complex at μM concentrations and rapidly accumulates in the nuclei of AML cells. We found that treatment of AML cells with MYBMIM, but not with its inactive near-isosteric analogue TG3, led to the displacement and dissociation of MYB:CBP complex in cells, causing rapid downregulation of MYB-dependent gene expression including MYC and BCL2 oncogenes. This was associated with obliteration of H3K27Ac-driven oncogenic enhancers induced by CBP and enriched for MYB binding sites (Figure 1A; p=1e-118). Both human MLL-rearranged and non-rearranged AML cells, but not normal CD34+ umbilical cord blood progenitor cells, underwent sustained mitochondrial apoptosis in response to MYBMIM treatment, an effect that could be partially blocked by ectopic expression of BCL2. We observed that MYBMIM treatment (50 mg/kg/day) impeded leukemia progression and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells (Figure 1B; p=3.8e-3). These findings emphasize the exquisite dependence of human AML on MYB:CBP transcriptional dysregulation, and establish a pharmacologic approach for its therapeutic blockade. Figure MYBMIM blocks oncogenic gene expression (A) and impedes leukemogenesis in vivo(B). Figure. MYBMIM blocks oncogenic gene expression (A) and impedes leukemogenesis in vivo(B). Disclosures Armstrong: Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.