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Human acyloxyacyl hydrolase is an unusual lipase, found in phagocytic cells, that removes acyl chains from bacterial lipopolysaccharides (LPS) and glycerolipids. It is a heterodimer in which two glycosylated peptides are linked by disulfide bonding. The large subunit contains the active site serine, while the smaller subunit has striking sequence similarity to the saposins, peptide cofactors for several sphingolipid hydrolases. Since rabbits and mice are widely used for studies of LPS—animal interactions, we asked if murine and lapine AOAHs resemble the human enzyme. We report here that murine and lapine AOAHs share the distinctive features of the human AOAH primary sequence and have similar affinity for LPS. The structure of this unusual lipase appears to have been highly conserved. more...

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia. Mol Cancer Ther; 12(12); 2675–84. ©2013 AACR. more...

The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC) using 6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45(+) cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection. more...

Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression. To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression. In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. Disclosures No relevant conflicts of interest to declare. more...

The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. Methods: B cell fractions- B cell progenitors from wild type mice were purified using flow cytometry. Gene Expression- RNA was collected and reverse transcriptase quantitative PCR was performed to quantitate Pax5, Etv6, Ebf1 and actin transcripts. Chromatin Immunoprecipitation- Cells were collected; proteins were cross-linked to DNA with formaldehyde, cell lysates were sonicated and immunoprecipitation was performed using 5μg indicated antibody or species-matched IgG as a negative control. DNA was recovered and assayed by quantitative PCR. Results: Recent studies have revealed a role for ETV6 germline mutations (P214L amino acid change) in the predisposition to Acute Lymphoblastic Leukemia (ALL). These mutations impair the transcriptional activity of ETV6 in a dominant negative fashion. Here, we demonstrate that Etv6 expression is inversely correlated to Pax5 and Ebf1 expression during B cell development (r2 = .9993; P = 0.0167). In a murine lymphoid progenitor line (Ba/F3), ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). In addition, Pax5 expression was increased by overexpression of EBF1, a known Pax5 transcriptional activator, in cells expressing the ETV6 P214L mutant (P≤0.05). This data suggests that loss of functional ETV6 is necessary for EBF1 to induce Pax5 expression. To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Aberrant expression of Pax5 leads to myeloid lineage skewing and an increase in biphenotypic and myeloid leukemias. Patients with ETV6 germline mutations have a higher percentage of monocytes compared to unaffected family members, which we hypothesize, is due to aberrant PAX5 expression (P = 0.008). Conclusions: ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. Citation Format: Courtney L. Jones, Courtney J. Fleenor, Seth Welsh, Leila Noetzli, Susan Fosmire, Dmitry Baturin, Jorge Di Paola, James R. Hagman, Christopher C. Porter. ETV6 represses Pax5 in early B-cell development. [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 2000. more...

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide short-hairpin RNA screens to identify proteins that mediate AML cell fate after cytarabine exposure; gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context; and examination of existing gene expression data from primary patient samples. Integration of these independent analyses strongly implicates cell-cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as a potential therapeutic target in AML. more...

We examined the presence of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) abnormalities that could contribute to the origin or progression of naturally occurring canine endothelial tumors (hemangiosarcoma). Our results document somatic point mutations or deletions encompassing the PTEN C-terminal domain in canine hemangiosarcoma that might provide cells a survival advantage within their microenvironment. This represents the first characterization of a naturally occurring, highly metastatic tumor with biologically significant mutations of PTEN in the C-terminal domain. more...

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (alpha(v)beta(3) integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular. more...

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas(+) melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery. more...

The calcineurin-regulated transcription factor, nuclear factor of activated T cells (NFAT), controls many aspects of T cell function. Here, we demonstrate that the calcineurin/NFAT pathway negatively regulates the expression of cyclin-dependent kinase 4 (CDK4). A canonical NFAT binding site was identified and found to be sensitive to calcium signals, FK506/CsA, and histone deacetylase activity and to not require AP-1. Ectopic expression of NFATc2 inhibited the basal activity of the human CDK4 promoter. Additionally, both calcineurin Aalpha(-/-) and NFATc2(-/-) mice had elevated protein levels of CDK4, confirming a negative regulatory role for the calcineurin/NFAT pathway. This pathway may thus regulate the expression of CDK4 at the transcriptional level and control how cells re-enter a resting, nonproliferative state. more...

Background: Survival rates for acute lymphoblastic leukemia (ALL) have risen dramatically but up to 20% of patients relapse and their prognoses are dismal. Resistance to glucocorticoid (GC) agonists is a hallmark of relapsed ALL and a strong predictor of outcome at diagnosis (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). However, the underlying biological pathways that mediate sensitivity to GCs remain to be determined. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cells. Methods: Genome-wide screening was performed using pooled shRNA libraries coupled with next generation sequencing to identify genes that when depleted promote GC sensitivity. Statistical analysis using Bioinformatics for Next Generation Sequencing (BiNGS) and Redundancy and Fold Change (RFC) were employed to identify candidate genes that mediate prednisolone sensitivity (Porter et. al, Leukemia 2012). Validation of hits from the primary screen were performed in Reh and RS4;11 cells. Knockdown of candidate genes MEK2 and MEK4 was determined by western blot. Changes in chemosensitivity upon MEK2 and MEK4 knockdown were determined by Cell Titer-Glo assay (Promega). The levels of apoptotic cells upon chemotherapy treatment in control and knockdown cell lines was determined by Annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA), followed by flow cytometry using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). The levels of downstream GC target genes including NR3C1, GILZ, and BIM were determined by RT-PCR. The levels of target proteins including GR, pERK, ERK, MEK1, and p53 were determined by western blot. pERK levels in primary matched pairs were determined by multiparameter phosphoflow cytometry. Results: In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with integrated analysis of relapse-specific genetic and epigenetic changes (Hogan et. al, Blood 2012) allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. Interestingly, depletion of MAPK pathway members, MEK2 and MEK4, increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to GCs by increasing the mRNA and protein levels of the glucocorticoid receptor (GR). This resulted in greater induction of the GR target genes GILZ and BIM upon prednisolone exposure over time. Importantly, depletion of MEK4 did not affect sensitivity of ALL cells to other chemotherapy agents (doxorubicin, etoposide, and 6-thioguanine). By contrast, MEK2 knockdown increased the sensitivity of cells to each of the chemotherapy agents tested including prednisolone, doxorubicin, etoposide, and 6-thioguanine. Depleting MEK2 decreased activated pERK and increased levels of p53. Over expression of a dominant negative p53 in MEK2 deficient cells reversed sensitivity to doxorubicin and prednisolone, indicating that MEK2 expression mediates chemosenstivity in a p53 dependent manner. Furthermore, inhibition of MEK1/2 pharmacologically with trametinib increased sensitivity of ALL cells to chemotherapy. Trametinib treatment also resulted in increased levels of p53. To determine if activation of the MAPK pathway in patients is associated with recurrent disease we examined seven matched diagnosis and relapse primary samples for MAPK activation as determined by pERK staining, and observed increased pERK levels at relapse in all samples tested. Conclusion: Our data indicate that activation of the MAPK pathway promotes chemoresistance and may drive the development of recurrent disease in pediatric ALL.Asdisrupting MEK2 and MEK4 sensitizes cells to chemotherapy, this makes the MAPK pathway an attractive target for therapeutic intervention in relapsed ALL. Disclosures No relevant conflicts of interest to declare. more...

Triple negative breast cancers (TNBCs) are among the most aggressive and heterogenous breast cancers characterized by a high propensity to invade, metastasize, and relapse; the exact mechanisms by which TNBCs mediate progression is unclear and thus, new insights into the biology of TNBC are desperately needed. We have developed a novel anti-cancer compound (AMPI-109) that is selectively efficacious against TNBC cell-lines. We conducted a genome-wide functional genomic shRNA screen to identify modifiers of AMPI-109 sensitivity and identified PTP4A3 (P3), a protein tyrosine phosphatase implicated in invasive, node positive TNBC tumors and associates with poor survival outcome, as a mediator of AMPI-109 action. In silico modeling predicted that AMPI-109 binds the catalytic site of P3 with favorable energetics. To investigate whether AMPI-109 could impact the activity of P3, we performed an in vitro phosphatase assay and demonstrated that AMPI-109 impaired the catalytic activity of P3. To investigate an oncogenic role of P3 in TNBC, we conducted stable lentiviral knockdown of P3 (ShP3) in BT-20 TNBC cells and demonstrated that loss of P3 leads to substantial growth inhibition (48%, p = 0.0055), similar in magnitude to AMPI-109 at 100 nM dose (66%, p = 0.00005). AMPI-109 also decreased P3 mRNA and protein levels. Accordingly, we blocked de novo protein synthesis and observed enhanced P3 protein degradation in the presences of AMPI-109. This degradation was reversible in the presence of the proteasome inhibitor MG-132, suggesting that AMPI-109 may induce P3 degradation through a proteasome-dependent mechanism. Functionally, high expression of P3 has been implicated in driving metastatic phenotypes in colorectal cancer but its role in TNBC cell migration has not been investigated. Wound-healing assays demonstrated that both AMPI-109 and ShP3 significantly impeded the migratory capacity of BT-20 cells (AMPI-109: 85% reduction, shP3: 80% reduction). AMPI-109 and ShP3 also conferred a higher rate of apoptosis induction as measured by activation of pro-apoptotic caspases-3 and 7 suggesting that the growth inhibitory phenotypes observed could potentially be attributed to increases in apoptotic cell death. To determine how P3 mechanistically exerts its oncogenic effect in the context of modulating cell signaling pathways, we carried out co-expression analysis of mRNAs that tracked with P3 expression across a suite of TNBC cell lines. We identified TRAPPC9, an activator of the NF-kappa B pathway, as correlating positively with P3 (r =0.652). Collectively, our results identify P3 as a target of AMPI-109 and that P3 has an oncogenic role in TNBC. Future studies will further characterize the relationship between AMPI-109, P3 and the NF-kappa B pathway. Furthermore, because of the specificity of AMPI-109 for TNBC cells, it may represent a new tool for understanding the molecular basis underlying the aggressive nature of this disease. Citation Format: Hamid H. Gari, Rahul Ray, Scott Lucia, Christopher C. Porter, Christy M. Gearheart, Susan Fosmire, Gregory D. DeGala, Zeying Fan, Yuanbin Ru, Ann D. Thor, James R. Lambert. PTP4A3 is oncogenic and modulates triple negative breast cancer growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4604. doi:10.1158/1538-7445.AM2014-4604 more...

While some patients with acute leukemia are cured, for many subsets of patients current therapeutic strategies are not adequate. Novel therapeutic approaches are needed for patients with higher risk leukemias, including T-ALL and AML. We and others identified Wee1 as a potential target in AML cells using RNAi screening. We have validated chemosensitization to cytarabine by genetic and pharmacologic inhibition of Wee1 in AML cell lines and primary patient samples ex vivo. A Wee1 inhibitor, MK1775, is in clinical development. We sought to further our findings with a wider range of conventional anti-leukemia agents, to determine whether the functionality of p53 influences chemosensitization, and to determine the tolerability and efficacy of MK1775 in combination with cytarabine in a mouse model of leukemia. We have found that MK1775 synergistically inhibits proliferation of the T-ALL Jurkat cell line with several antimetabolite chemotherapeutics including cytarabine, 6-thioguanine, and methotrexate. In contrast, MK1775 does not sensitize Jurkats to doxorubicin or etoposide, suggesting specific sensitization to antimetabolites. The addition of MK1775 enhances the antimetabolite induced apoptosis, as measured by Annexin V/7-AAD staining, and PARP cleavage measured by Western blotting. As expected, the addition of MK1775 enhances DNA damage induced by cytarabine as measured by γH2AX staining and flow cytometry, although preliminary data suggest that this is not the only mechanism of enhanced cell death, as a substantial proportion of cleaved PARP+ cells does not stain for γH2AX. In addition, we have found that AML cell lines with both wild-type and mutated TP53 are sensitive to chemosensitization by Wee1 inhibition. Furthermore, in isogenic models of p53 dysfunction, we have found that the functionality of p53 does not influence chemosensitization. Lastly, in an aggressive mouse model of AML, we observed enhanced disease control and survival in mice treated with MK1775 and ARA-C as compared to ARA-C alone. Hematotoxicity associated with treatment was related to the duration of combination therapy, but was tolerated well with intermittent dosing. Taken together, these data indicate that Wee1 inhibition may enhance the efficacy of several clinically relevant anti-leukemia agents, particularly the antimetabolites, but not topoisomerase inhibitors. Further, they suggest caution about the use of p53 mutation as a biomarker predictive of response to Wee1 inhibition. Moreover, we show that the addition of MK1775 to cytarabine is tolerable and more effective than cytarabine alone in vivo. Ongoing studies are aimed at better understanding the mechanism of combinatorial effect and to determine whether combination therapy is more efficacious than single agent therapy in xenograft models of leukemia. These data provide justification for early phase clinical trials of MK1775 in combination with antimetabolites in patients with high risk acute leukemia. Disclosures: No relevant conflicts of interest to declare. more...

Abstract 3503 Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge of the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to specific molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational strategies, we performed genome-wide shRNA screens to identify proteins which mediate AML cell fate in cytarabine in 2 AML cell lines (Molm13 and MV4-11). Over 55,000 shRNAs targeting over 11,000 genes were quantified by deep sequencing to identify shRNAs under-represented in the context of cytarabine as compared to no treatment. Complementary analyses identified 125 genes as mediators of AML cell fate in cytarabine. In addition we performed gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context and examined existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell fate in cytarabine. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival in cytarabine. Pharmacologic inhibition of WEE1 in several, but not all, AML cell lines is synergistic with cytarabine, suggesting underlying molecular susceptibility to this combination of drugs. A WEE1 inhibitor is in Phase II trials in solid tumors, primarily as a means to abrogate the G2/M checkpoint in tumors with TP53 dysfunction. Further experiments demonstrate that inhibition of WEE1 prevents slowed S-phase progression induced by cytarabine in AML cells, broadening the functions of WEE1 that may be exploited therapeutically. Preliminary experiments indicate synergistic inhibition of AML cellular proliferation with daunorubicin in some AML cell lines. Experiments to determine whether WEE1 inhibition in combination with chemotherapy prolongs survival of mice with leukemia are underway. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. Disclosures: No relevant conflicts of interest to declare. more...

Background The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities. Results Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Conclusion Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories. more...

A panel of documented arthritogenic Shigella flexneri strains as well as an epidemic-associated non-arthritogenic Shigella sonnei control was used to identify a 2-megadalton plasmid specific to the arthritogenic strains. The plasmid, pHS-2, contains a DNA sequence that encodes a 22-amino acid polypeptide encompassing a pentapeptide homologous to part of the polymorphic region of the alpha-1 domain of HLA-B27. These results suggest that molecular mimicry between arthritogenic bacterial-encoded epitopes and the HLA-B27 molecule may play a role in the development of reactive arthritis. more...

The development of reactive arthritis, a sterile inflammatory polyarthropathy that primarily affects HLA-B27 positive individuals, has been associated with previous enteric infections caused by various gram-negative bacteria. The possibility that a common bacterial epitope triggers the disease was investigated by screening a panel of documented arthritogenic Shigella strains as well as 2 epidemic-associated nonarthritogenic Shigella controls. A 2-Md plasmid specific to the arthritogenic strains was identified and sequenced. The plasmid encodes a number of small peptides that could be related to the development of reactive arthritis. Within 1 of these is a stretch of 5 consecutive amino acids, inferred from the DNA sequence and contained within an open reading frame, that is homologous to amino acid residues 71-75 of the polymorphic region of the alpha 1 domain of HLA-B27. The data indicate that there is a bacterial plasmid common to arthritogenic Shigella strains that may play a role in triggering reactive arthritis. The finding that this plasmid encodes an epitope shared with HLA-B27 suggests that molecular mimicry may play a role in the induction of this disease. more...

The role of tumor suppressor genes in the pathogenesis of canine melanoma is incompletely understood. The genes encoding the tumor suppressors p53, Rb, p21 ( waf-1), p16 ( ink-4a), and PTEN have been postulated to contribute to the pathogenesis of melanoma in humans and experimental animal models. To assess whether inactivation of these genes similarly contributes to the origin and progression of canine melanoma, we examined their expression in seven distinct canine melanoma cell lines and in 31 retrospective samples (representing 29 dogs) of spontaneous canine melanoma. Various patterns suggestive of loss of tumor suppressor function emerged in these cell lines. The most frequently observed abnormality was loss or significant reduction of p16 expression in six of seven cell lines and in 21 of 26 tumor samples. Loss or significant reduction of PTEN expression was seen in four of seven cell lines and in 13 of 27 tumor samples. Although p53 was detectable in all the cell lines and in 24 of 30 tumors, exclusion of p53 from the nuclear compartment was observed in each of the cell lines and in 18 of 25 tumor samples. These results indicate that loss of function of these tumor suppressor proteins is a common occurrence that may contribute to the origin of canine melanoma. In our sample population, abnormalities in the expression or localization of one or more tumor suppressor proteins occurred with similar frequency in malignant and benign tumors; thus, additional work is necessary to determine how these proteins may impact disease progression and response to therapy. more...

Background The etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease. Methods We first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR. Results Each of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma). Conclusions The data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells. more...