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ObjectiveIn epidemiological and experimental research, high folic acid intake has been demonstrated to accelerate tumor development among populations with genetic and/or molecular susceptibility to cancer. Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder predisposing affected individuals to tumorigenesis, including benign plexiform neurofibromas; however, understanding of factors associated with tumor risk in NF1 patients is limited. Therefore, we investigated whether pregestational folic acid intake modified plexiform-like peripheral nerve sheath tumor risk in a transgenic NF1 murine model.ResultsWe observed no significant differences in overall survival according to folate group. Relative to controls (180 days), median survival did not statistically differ in deficient (174 days,P=0.56) or supplemented (177 days,P=0.13) folate groups. Dietary folate intake was positively associated with RBC folate levels at weaning, (P=0.023, 0.0096, and 0.0006 for deficient vs. control, control vs. supplemented, and deficient vs. supplemented groups, respectively). Dorsal root ganglia (DRG), brachial plexi, and sciatic nerves were assessed according to folate group. Mice in the folate deficient group had significantly more enlarged DRG relative to controls (P=0.044), but no other groups statistically differed. No significant differences for brachial plexi or sciatic nerve enlargement were observed according to folate status.

Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow–transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit+/lineage−/Sca-1+ (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.

Summary Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndrome (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. Although homozygous knockout of Hspa9 is embryonically lethal, mice with heterozygous deletion of Hspa9 (Hspa9(+/-)) are viable and have a 50% reduction in Hspa9 expression. Hspa9(+/-) mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9(+/-) mice have a cell-intrinsic reduction in bone marrow colony-forming unit-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9(+/-) B-cell progenitors. We further reduced Hspa9 expression (

This abstract is being presented as a short talk. A full abstract is printed in the Proffered Abstracts section (PR06) of the Conference Proceedings. Citation Format: Cara Lunn Shirai, James N. Ley, Brian White, Justin Tibbitts, Jin Shao, Matthew Ndonwi, Sanghyun Kim, Theresa Okeyo-Owuor, Timothy A. Graubert, Matthew J. Walter. Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B41.

Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3'-splice site recognition, in 11% of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased frequency of Annexin V+ staining (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency in bone marrow and spleen of mice. U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+ Lineage - (KL) cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for KLS (c-Kit+ Lineage- Sca-1+) cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as an increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice at 5 days is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to controls (p Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice. We identified 402 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) (FDR 2 |log2| fold change). We also observed a preference of the mutant U2AF1(S34F) to skip exons (n=72) and alternative splice sites (n=45) with a T in the -3 position relative to the AG splice acceptor site. We intersected our junction results with RNA sequencing from AML patient samples with U2AF1 mutations and human CD34+ cells expressing U2AF1(S34F), and we identified homologous dysregulated junctions in all 3 datasets that are currently being tested for their functional significance. Together, these results suggest that mutant U2AF1 expression contributes to the altered pre-mRNA splicing and hematopoiesis observed in patients with U2AF1 mutations. Citation Format: Cara Lunn Shirai, James N. Ley, Brian White, Justin Tibbitts, Jin Shao, Matthew Ndonwi, Sanghyun Kim, Theresa Okeyo-Owuor, Timothy A. Graubert, Matthew J. Walter. Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR06.

Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type recipient mice prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Mutations in 8 spliceosome genes have been identified in up to half of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3’-splice site recognition, in 11% of MDS patients. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis, we created site-specific, single-copy, doxycycline-inducible (controlled by the reverse tetracycline-transactivator [rtTA] transgene) U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to doxycycline induction of transgene expression. We observed a doxycycline dose-dependent increase in expression of U2AF1(S34F) and U2AF1(WT) transgenes by RT-PCR and pyrosequencing analysis, and we subsequently chose a dose that induces equivalent levels of exogenous U2AF1 transgene to endogenous mouse U2af1 transcripts. Following 4 weeks of doxycycline induction of transgene expression in vivo, U2AF1(S34F)-recipient mouse bone marrow cells showed increased alternative splicing of endogenous Fmr1 pre-mRNA, a transcript previously shown to be alternatively-spliced in U2AF1 mutant MDS patient samples, when compared to U2AF1(WT)- and rtTA only-recipient mouse bone marrow cells (p Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice display a decrease in WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (average 4.5 vs 6.7 and 7.1 K/µl, respectively; p≤0.01). However, there are no significant differences in bone marrow cellularity or spleen size in these mice (n=9-11). U2AF1(S34F)-recipient mice have reduced frequency of peripheral blood monocytes (average 1.2 vs 3.2 and 2.6%, p In addition, 4 weeks of U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency. U2AF1(S34F)-recipient mice have approximately a 1.5-fold increase in the frequency of progenitor cells in their bone marrow measured by flow cytometry (linlow/c-kit+/Sca-1-) (average 4.1 vs 2.6 and 2.9%; p Collectively, these data indicate that mutant U2AF1(S34F) expression in mouse bone marrow cells results in altered pre-mRNA splicing and changes in hematopoietic cell lineage distribution, as well as increased bone marrow and spleen progenitor and stem cell populations. Furthermore, these results suggest that mutant U2AF1 may contribute to altered hematopoiesis in patients. Disclosures: No relevant conflicts of interest to declare.

Myeloid and erythroid differentiation defects and cytopenias are most commonly described in myelodysplastic syndromes (MDS), however, a reduction in B-cell progenitors exists. The genetic events contributing to this reduction are poorly understood. Interstitial deletion or loss of one copy of the long arm of chromosome 5 (del5q) is the most common cytogenetic abnormality associated with MDS. Two commonly deleted regions on del(5q) have been described and no biallelic mutations have been identified implicating haploinsufficiency of genes on this interval as a driving mechanism. We, and others, have identified several del(5q) candidate genes, including RPS14, EGR1, CTNNA1, APC, NPM1, DIAPH1, miR145, miR146a, and HSPA9. Consistent with haploinsufficiency, HSPA9 mRNA levels are 50% reduced in del(5q) patients. We previously showed that knockdown of Hspa9by shRNA in a murine bone marrow transplant model resulted in a significant reduction in murine B-cells in the bone marrow, spleen and peripheral blood. To further characterize the role of Hspa9 in hematopoiesis, we created Hspa9 heterozygous mice (Hspa9+/-). Heterozygotes express 50% less Hspa9 protein and are born at normal Mendelian frequencies (N>100). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, Hspa9+/- mice show a significant reduction in CFU-PreB colonies compared to their wild-type littermates, indicating B-cell progenitor defects (14 vs. 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p Despite the reduction in CFU-PreB colony numbers, frequencies of freshly isolated early B-cell progenitor and precursor populations in the bone marrow and spleen of Hspa9+/- mice are not different than wild-type littermate controls when assessed by flow cytometry (common lymphoid progenitor, Hardy fractions A-F). We hypothesized that these mice were able to compensate for B-cell alterations caused by loss of Hspa9 in vivo. Consistent with our hypothesis, the reduction in CFU-PreB colony numbers was partially rescued by increasing the concentration of IL-7 in the media. Hspa9+/- colony numbers increased 1.8 fold when the IL-7 concentration was increased from 10ng/mL to 50ng/mL compared to 0.80 fold for wild-type littermates (p=0.03, N=6 mice/genotype). This effect was unique to IL-7. Adding increasing concentrations of Flt-3 ligand, another cytokine that contributes to early B-cell development, did not alter CFU-PreB colony formation. We isolated B220+ cells from Day 7 CFU-PreB cultures for gene expression array analysis and observe reduced expression of genes promoting B-cell proliferation and activation in Hspa9+/- compared to Hspa9+/+ cells. Since IL-7 is the only supportive cytokine in the methylcellulose media, can partially rescue the reduced CFU-PreB phenotype, and is required for early B-cell development and survival, we hypothesized that Hspa9 haploinsufficiency inhibits transduction of IL-7 signaling. We tested this hypothesis using an IL-7 dependent mouse B-cell line (B7 cells; Ba/F3 cells that stably express the IL-7 receptor). Knockdown of Hspa9 by siRNAs resulted in a 8-fold reduction in cell number after 4 days in culture (p=0.004, confirmed with two independent siRNAs) and was associated with an increase in apoptosis and reduction in cells in S-phase of the cell cycle. Knockdown of Hspa9 in B7 cells resulted in reduced levels of phosphorylated Stat5, an immediate downstream target of IL-7 receptor stimulation, compared to cells treated with a non-targeting siRNA (measured at 5, 10, 15 and 30 minutes following 10ng/mL IL-7 stimulation, p≤0.03). Ongoing studies will further interrogate the effects of Hsap9 knockdown on Jak-Stat signaling. Collectively, these data implicate that loss of HSPA9 alters IL-7 signaling, potentially contributing to the reduction of B-cell progenitors observed in patients with del(5q)-associated MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract 553 Myelodysplastic syndromes (MDS) are stem cell disorders characterized by ineffective hematopoiesis with increased levels of hematopoietic cell apoptosis. Recent discoveries by our group and others suggest that perturbations in pre-mRNA splicing may play a role in MDS pathogenesis. Indeed, more than half of all MDS patients have a mutation in one of eight splicing factors. U2AF1 (U2AF35), a gene encoding a splicing factor involved in intronic 3'-splice site recognition, is mutated in 8.7% of MDS patients. The consequence of the highly recurrent serine to phenylalanine mutation at position 34 (S34F) of U2AF1 in hematopoiesis is unknown. Therefore, to examine the effects of mutant U2AF1(S34F) on hematopoiesis, we utilized the MSCV-IRES-GFP retroviral system to introduce mutant U2AF1(S34F) or wild type U2AF1(WT), or an empty vector control, into mouse bone marrow cells for in vitro and in vivo studies. Expression of U2AF1(S34F) results in reduced expansion of transduced bone marrow cells (marked by GFP) compared to both U2AF1(WT) or empty vector-transduced cells grown in suspension culture (2 vs 4-fold change, respectively; p To overcome some of the limitations of retroviral models, we created a single-copy, doxycycline-inducible U2AF1(S34F) transgenic mouse to model the effect of U2AF1(S34F) expression on hematopoiesis. As a control for U2AF1 protein overexpression, we created an additional single-copy, doxycycline-inducible U2AF1(WT) transgenic mouse with transgene integration into the same locus as the U2AF1(S34F) mouse. Induction of U2AF1(S34F) transgene expression in bone marrow cells in culture with doxycycline treatment (200 ng/ml for 5 days) resulted in reduced cell numbers when compared to uninduced U2AF1(S34F) transgenic cells (ratio of growth of induced/uninduced cells = 0.38), while cell proliferation was not altered for U2AF1(WT) transgenic cells (ratio of growth of induced/uninduced cells = 1.13) (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1714 Patients with myelodysplastic syndromes (MDS) have a clonal hematopoietic stem cell disorder that results in dysplastic hematopoietic cells in their bone marrow as well as peripheral blood cytopenias. In addition to the commonly described erythroid and myeloid differentiation defects associated with MDS, a reduction in bone marrow B-cell progenitors exists in patients. The genetic events contributing to the reduction in B-cell progenitors remain poorly understood. The most common cytogentic abnormality identified in patients with MDS, occurring in approximately 35% of patients, is heterozygous interstitial deletion or loss of the long arm of chromosome 5 (5q). The interstitial deletions on chromosome 5 are single copy losses, and no biallelic disruptions of genes in deleted regions have been identified, implicating haploinsufficiency as the underlying genetic mechanism. We, and others, have shown that the levels of HSPA9 mRNA expression are reduced ∼50% in patients with del(5q) when compared to MDS patients without del(5q), consistent with a haploinsufficient phenotype. To model haploinsufficiency, we used shRNA to achieve ∼50% knockdown of Hspa9 in a murine bone marrow transplant model. This model showed a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, implicating HSPA9 haploinsufficiency may contribute to the B-cell alterations observed in MDS patients with del(5q). To further evaluate HSPA9 haploinsufficiency in vivo, we created a mouse model with a heterozygous deletion of Hspa9 (Hspa9+/−) and confirmed a 50% reduction in Hspa9 protein levels in bone marrow and spleen of these mice by Western blot. Hspa9+/− mice are born at normal Mendelian frequencies (N>100), however, breeding heterozygous mice suggests Hspa9−/− mice are embryonic lethal (24 Hspa9+/+:38 Hspa9+/−:0 Hspa9−/−). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, the numbers of bone marrow CFU-preB colonies as assessed by methylcellulose assay, are significantly reduced in Hspa9+/− mice compared to Hspa9+/+ littermates (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p Total frequencies of common lymphoid progenitors and B-cell precursors (Hardy fractions A, B/C, D, E and F) as assessed by flow cytometry are no different in Hspa9+/− and Hspa9+/+ mice. Therefore, we hypothesize that early Hspa9+/− B-cells may have an intrinsic signaling defect which can be compensated for in vivo. Early B-cell maturation is dependent on intracellular signaling mediated through cell surface receptors in response to environmental cytokines. Consistent with our hypothesis, we showed that Hspa9+/− CFU-preB in vitro colony formation is partially rescued by increasing concentrations of IL7 while Hspa9+/+ colony numbers remain unchanged (fold change in colony formation from 10ng/mL to 50ng/mL IL7 was 1.80 for Hspa9+/− vs. 0.80 for Hspa9+/+, p=0.03, N=6 mice/genotype). Supplementation of the media with another cytokine that contributes to early B-cell maturation, Flt3 ligand, does not alter Hspa9+/− or Hspa9+/+ CFU-preB colony formation, further implicating altered IL7 signaling. We are currently investigating the downstream responses to IL7 stimulation in B-cell progenitors from Hspa9+/− mice. Collectively, these data implicate loss of HSPA9 as a contributing factor in the reduction of B-cell progenitors observed in patients with del(5q) associated MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract 3829 Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and an increased risk of acute myeloid leukemia (AML) development. In addition to erythroid and myeloid differentiation defects in MDS, a reduction in bone marrow B-cell progenitors exists in MDS patients. The genetic events leading to the development of MDS, reduction in B-cell progenitors, and the subsequent transformation to AML are poorly understood. Heterozygous, interstitial deletions or loss of the long arm of chromosome 5 (5q) are among the most commonly acquired genetic abnormalities found in MDS, occurring in approximately 35% of patients. Within the commonly deleted regions of chromosome 5 are several genes implicated in MDS/AML development including RPS14, EGR1, CTNNA1, and HSPA9. HSPA9 mRNA expression levels have been shown to be 50% reduced in patients with deletions of the common region on chromosome 5, consistent with a haploinsufficient level of expression. shRNA knockdown of Hspa9 in a murine bone marrow transplant model resulted in a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, suggesting Hspa9 haploinsufficiency may contribute to B-cell alterations observed in MDS patients with chromosome 5 deletions. In order to further test whether HSPA9 haploinsufficiency alters hematopoiesis in vivo, we created an Hspa9 heterozygous knockout mouse (Hspa9+/−). Hspa9+/− mice were born at normal Mendelian frequencies (N>100) and expressed 50% less protein in their spleen and bone marrow compared to wildtype littermates by Western blot. Breeding heterozygous mice yielded 24 Hspa9+/+, 38 Hspa9+/−, and 0 Hspa9−/− mice, suggesting that homozygous knockout mice are embryonic lethal. Basal hematopoiesis in the bone marrow, spleen, and peripheral blood of Hspa9+/− mice is normal at 2 (N=3–10 mice/genotype) and 6 (N=5 mice/genotype) months of age. Complete blood counts, flow cytometric analysis of lineage markers (B220, CD3e, Gr1, CD115, CD71, and Ter119), and hematopoietic organ cellularity do not differ between Hspa9+/− and Hspa9+/+ littermate controls. However, beginning at 2 months of age, Hspa9+/− mice have a significant reduction in the number of pre B-cell progenitors compared to Hspa9+/+ littermate control mice using methylcellulose cultures (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p In addition to the reduction in B-cell progenitors in MDS, recent gene expression profiling results indicate that del(5q) is specifically associated with a reduced expression of B-cell signaling genes in CD34+ MDS progenitor cells. Two of the most highly down-regulated gene sets in del(5q) MDS CD34+ cells compared to normal CD34+ cells included B-cell signaling genes (Leukemia. 2010 Apr;24(4):756–64.). Using independent Affymetrix U133plus2 gene expression profiling data from our institution, we confirm that the most down-regulated genes in CD34+ progenitor cells from del(5q) or non-del(5q) MDS patients compared to control CD34+ cells are also B-cell development genes using Gene Set Enrichment Analysis (GSEA)(p Collectively, the data suggest that loss of HSPA9 may contribute to the reduction of B-cell progenitors observed in patients with del(5q) associated MDS. Follow-up analysis of basal and stress hematopoiesis in aged Hspa9+/− mice are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

Midkine (MDK), a heparin-binding growth factor, is highly expressed in pediatric ALL, neuroblastomas, and various solid tumors. Previous gene expression profiling studies conducted in our laboratory on a cohort of 220 children with B precursor ALL treated on Children’s Oncology Group (COG) clinical trials have shown that higher levels of MDK expression in leukemic blasts are strongly associated with continuous complete remission (CCR) and that MDK is a powerful prognostic predictor of outcome (Wilcoxon ranksum test, p=0.000001), particularly in patients with central nervous system (CNS) involvement (Wilcoxon ranksum test, p =0.009). To explore the role of MDK in hematopoiesis and leukemogenesis and to further explore MDK as a potential novel target for ALL therapy, we examined MDK mRNA expression in three stages of normal B cell development in healthy controls and compared it to expression levels in leukemic blasts from children with B-ALL. Quantitative RT-PCR assays showed that MDK mRNA levels in CD19+ cells from peripheral blood mononuclear cells (PBMNC) were significantly higher than those in CD34+ or CD19+ cells from bone marrow (BM) (Mann Whitney test, p=0.0025 and 0.0018, respectively). ALL leukemic blasts samples expressed significantly higher levels of MDK than those in CD19+ cell in the BM of healthy controls (Mann Whitney test, p=0.0043). Similar results were obtained by ELISA detection of MDK protein in plasma of ALL patients and healthy controls. We have also investigated the relationship of MDK expression and the chemo-sensitivity of ALL cell lines. Using a lentiviral system, we have successfully over-expressed or knocked down MDK gene expression in the REH cell line containing the t(12;21). Unexpectedly, MTS assays revealed that MDK expression is inversely associated with sensitivity to L-asparaginase and doxorubicin, but not to vincristine and cytarabine, in REH cells. Higher levels of MDK were associated with chemo-resistance. Finally, we tested the effects of MDK expression on proliferation and apoptosis in REH cells and determined that although MDK expression was not associated with REH cell proliferation in vitro, it protected REH cells from doxorubicin-induced apoptotic cell death. Taken together, these results suggest that expression levels of MDK in ALL is inversely associated with in vitro drug sensitivity to specific anti-leukemic agents; consistent with observations in solid tumor models. It has been suggested that 4β1- and 6β1-integrins bind directly to MDK and are involved in MK-dependent cell migration. Further studies of the role of MDK signaling in vivo in the complex marrow/adhesion microenvironment and the effects on ALL survival and chemo-sensitivity and resistance are in progress.