Proviral Integration Site Research Articles

Activity of PIM Kinases in Chronic Lymphocytic Leukemia Modulates Tumor Cell Survival and Stromal Interactions through a Pleiotropic Mechanism Involving Modulation of CXCR4 - mTOR Pathway

The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells.In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p<0.001). PIM2 expression was higher in patients with advanced disease (Rai 3-4, p=0.004). Higher PIM2 expression was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood (p=0.024 and p=0.0059, respectively; Herishanu et al., 2010), suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of PIM kinases in these processes, we assessed whether PIM inhibition interferes with CXCR4 - mediated signaling and migration. We incubated primary CLL cells with a novel pan-PIM inhibitor SEL24-B489 and found decreased phospho-CXCR4 (Ser339) level and decreased CXCR4 surface expression. Primary CLL cells incubated with with SDF1 (500 ng/ml, 15-60 min) exhibited highly increased phosphorylations of mTOR (Ser2448) and Akt (Ser437). Since PIM kinases modulate mTOR signaling, we further investigated whether inhibition of PIM kinases with SEL24-B489 interferes with CXCR4/mTOR pathway. SEL24-B489 blocked baseline phosphorylation level of mTOR negative regulator p-TSC2 (Ser1798). Since TSC2 Ser1798 phosphorylation relieves its suppression on RHEB and promotes mTOR activity, we next assessed PIM inhibitor - induced changes in p-mTOR (Ser2448). Upon SEL24-B489 treatment, mTOR Ser2448 phosphorylation and activity of mTOR downstream substrates (p-Akt Ser473 and p-4EBP1 Thr37/46) markedly decreased. Pre-incubation of CLL cells with 10 uM SEL24-B489 or 10 uM mTOR inhibitor OSI-027 before SDF1 treatment restrained the increase of p-mTOR (Ser2448), p-Akt (Ser473), p-4EBP1 (Thr37/46) and p-TSC2 (Ser1798), and consequently impaired CLL cells migration in the SDF1 gradient. In 4 out of 7 analyzed patients the effect of SEL24-B489 on CLL migration was stronger than the effect of OSI-027 and in the remaining 3 patients, the effects of both inhibitors were similar. Since interactions of CLL cells with their microenvironment block the cytotoxicity of chemotherapeutic agents, we next compared the apoptotic response to SEL24-B489 in CLL cells cultured in the absence or presence of human bone marrow HS5 cells. CLL cells were seeded on HS5 monolayers and treated with SEL24-B489 (5 uM and 10 uM) for 48 hours. In 6 out of 7 cases SEL24-B489 overcame the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.Taken together, our data demonstrate that CXCR4/SDF1 signal in chronic lymphocytic leukemia cells is transduced through mTOR pathway and that CXCR4 - triggered mTOR activity is modulated by PIM kinases. Pan-PIM inhibitor SEL24-B489 decreased CXCR4 surface expression and SDF-1 - triggered mTOR activity. Finally, SEL24-B489 decreased protective effects of tumor microenvironment and induced CLL cells apoptosis even in the presence of stromal cells. Since overexpression of PIM kinases might be associated with adverse clinical characteristics at diagnosis, PIM inhibition might be a rational therapeutic strategy in CLL. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

The novel anti-adipogenic effect and mechanisms of action of SGI-1776, a Pim-specific inhibitor, in 3T3-L1 adipocytes.

The proviral integration site for moloney murine leukemia virus (Pim) kinases, consisting of Pim-1, Pim-2 and Pim-3, belongs to a family of serine/threonine kinases that are involved in controlling cell growth and differentiation. Pim kinases are emerging as important mediators of adipocyte differentiation. SGI-1776, an inhibitor of Pim kinases, is widely used to assess the physiological roles of Pim kinases, particularly cell functions. In the present study, we examined the effects of SGI-1776 on adipogenesis. The anti‑adipogenic effect of SGI‑1776 was measured by Oil Red O staining and AdipoRed assays. The effect of SGI‑1776 on the growth of 3T3‑L1 adipocytes was determined by cell count analysis. The effects of SGI‑1776 on the protein and mRNA expression of adipogenesis-related proteins and adipokines in 3T3‑L1 adipocytes were also evaluated by western blot analysis and RT‑PCR, respectively. Notably, SGI-1776 markedly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On a mechanistic level, SGI-1776 inhibited not only the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ) and fatty acid synthase (FAS), but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Moreover, SGI-1776 decreased the expression of adipokines, including the expression of leptin and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. These findings demonstrate that SGI-1776 inhibits adipogenesis by downregulating the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS and STAT-3.

HTLV-1 drives vigorous clonal expansion of infected CD8(+) T cells in natural infection.

BackgroundHuman T-lymphotropic Virus Type I (HTLV-1) is a retrovirus that persistently infects 5–10 million individuals worldwide and causes disabling or fatal inflammatory and malignant diseases. The majority of the HTLV-1 proviral load is found in CD4+ T cells, and the phenotype of adult T cell leukemia (ATL) is typically CD4+. HTLV-1 also infects CD8+ cells in vivo, but the relative abundance and clonal composition of the two infected subpopulations have not been studied. We used a high-throughput DNA sequencing protocol to map and quantify HTLV-1 proviral integration sites in separated populations of CD4+ cells, CD8+ cells and unsorted peripheral blood mononuclear cells from 12 HTLV-1-infected individuals.ResultsWe show that the infected CD8+ cells constitute a median of 5 % of the HTLV-1 proviral load. However, HTLV-1-infected CD8+ clones undergo much greater oligoclonal proliferation than the infected CD4+ clones in infected individuals, regardless of disease manifestation. The CD8+ clones are over-represented among the most abundant clones in the blood and are redetected even after several years.ConclusionsWe conclude that although they make up only 5 % of the proviral load, the HTLV-1-infected CD8+ T-cells make a major impact on the clonal composition of HTLV-1-infected cells in the blood. The greater degree of oligoclonal expansion observed in the infected CD8+ T cells, contrasts with the CD4+ phenotype of ATL; cases of CD8+ adult T-cell leukaemia/lymphoma are rare. This work is consistent with growing evidence that oligoclonal expansion of HTLV-1-infected cells is not sufficient for malignant transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0221-1) contains supplementary material, which is available to authorized users.

Lentiviral vector-mediated insertional mutagenesis screen identifies genes that influence androgen independent prostate cancer progression and predict clinical outcome.

Prostate cancer (PC) is the second leading cause of cancer related deaths in US men. Androgen deprivation therapy (ADT) improves clinical outcome, but tumors often recur and progress to androgen independent prostate cancer (AIPC) which no longer responds to ADT. The progression to AIPC is due to genetic alterations that allow PC cancer cells to grow in the absence of androgen. Here we performed an insertional mutagenesis screen using a replication-incompetent lentiviral vector (LV) to identify the genes that promote AIPC in an orthotopic mouse model. Androgen sensitive PC cells, LNCaP, were mutagenized with LV and injected into the prostate of male mice. After tumor development, mice were castrated to select for cells that proliferate in the absence of androgen. Proviral integration sites and nearby dysregulated genes were identified in tumors developed in an androgen deficient environment. Using publically available datasets, the expression of these candidate androgen independence genes in human PC tissues were analyzed. A total of 11 promising candidate AIPC genes were identified: GLYATL1, FLNA, OBSCN, STRA13, WHSC1, ARFGAP3, KDM2A, FAM83H, CLDN7, CNOT6, and B3GNT9. Seven out the 11 candidate genes; GLYATL1, OBSCN, STRA13, KDM2A, FAM83H, CNOT6, and B3GNT6, have not been previously implicated in PC. An in vitro clonogenic assay showed that knockdown of KDM2A, FAM83H, and GLYATL1 genes significantly inhibited the colony forming ability of LNCaP cells. Additionally, we showed that a combination of four genes, OBSCN, FAM83H, CLDN7, and ARFGAP3 could significantly predicted the recurrence risk in PC patients after prostatectomy (P = 5.3 × 10-5 ). © 2015 Wiley Periodicals, Inc.

A novel gammaretroviral shuttle vector insertional mutagenesis screen identifies SHARPIN as a breast cancer metastasis gene and prognostic biomarker.

Breast cancer (BC) is the second leading cause of malignancy among U.S. women. Metastasis results in a poor prognosis and increased mortality, but the molecular mechanisms by which metastatic tumors occur are not well understood. Identifying the genes that drive the metastatic process could provide targets for improved therapy and biomarkers to improve BC patient outcomes. Using a forward mutagenesis screen, BC cells mutagenized with a replication-incompetent gammaretroviral vector (γRV) were xenotransplanted into the mammary fat pad of immunodeficient mice. In this approach the vector provirus dysregulates nearby genes, providing a selective advantage to transduced cells to form metastases. Metastatic tumors were analyzed for proviral integration sites to identify nearby candidate metastasis genes. The γRV has a transgene cassette that allows for rescue in bacteria and rapid identification of vector integration sites. Using this approach, we identified the previously described metastasis gene WWTR1 (TAZ), and three other novel candidate metastasis genes including SHARPIN. SHARPIN was independently validated in vivo as a BC metastasis gene. Analysis of patient data showed that SHARPIN expression predicts metastasis-free survival after adjuvant therapy. Our approach has broad potential to identify genes involved in oncogenic processes for BC and other cancers. We show here it can identify both known (WWTR1) and novel (SHARPIN) BC metastasis genes.

The novel combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in acute myeloid leukemia via HSF pathway suppression.

Mammalian target of rapamycin (mTOR) signaling is a critical pathway in the biology of acute myeloid leukemia (AML). Proviral integration site for moloney murine leukemia virus (PIM) serine/threonine kinase signaling takes part in various pathways exerting tumorigenic properties. We hypothesized that the combination of a PIM kinase inhibitor with an mTOR inhibitor might have complementary growth-inhibitory effects against AML. The simultaneous inhibition of the PIM kinase by pan-PIM inhibitor AZD1208 and of mTOR by selective mTORC1/2 dual inhibitor AZD2014 exerted anticancer properties in AML cell lines and in cells derived from primary AML samples with or without supportive stromal cell co-culture, leading to suppressed proliferation and increased apoptosis. The combination of AZD1208 and AZD2014 rapidly activated AMPKα, a negative regulator of translation machinery through mTORC1/2 signaling in AML cells; profoundly inhibited AKT and 4EBP1 activation; and suppressed polysome formation. Inhibition of both mTOR and PIM counteracted induction of heat-shock family proteins, uncovering the master negative regulation of heat shock factor 1 (HSF1), the dominant transcription factor controlling cellular stress responses. The novel combination of the dual mTOR inhibitor and pan-PIM inhibitor synergistically inhibited AML growth by effectively reducing protein synthesis through heat shock factor pathway suppression.

The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.

Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study

The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

Improving the methodology for the detection of proviral integration sites in the host genome via high throughput sequencing

Bovine Leukemia Virus (BLV) and the closely related Human T-cell leukemia virus-1 (HTLV-1) are deltaretrovirus that induce leukemia/lymphoma in about ~5% of infected individuals. The mechanisms responsible for cellular transformation have remained largely enigmatic as both viruses are largely transcriptionally silent in tumors and show multiple integration sites in the host genome. The recent application of high throughput sequencing to track proviral insertion sites in the host genome has provided a number of insights into the evolution of deltaretrovirus infections and the progression of tumor clones in deltaretrovirus induced leukemia/lymphoma. However the protocols currently utilised have a number of limitations, including relatively high sequencing costs, the use of custom sequencing primers, no examination of the region upstream of the provirus and limited dynamic range for determining clone abundance. We have developed an alternative high throughput sequencing protocol for identifying proviral integration sites in BLV and HTLV-1 infected individuals that uses off-the-shelf Illumina primers for the addition of adapters and indexes. This greatly simplifies the process of multiplexing libraries and does away with the need for custom sequencing primers. Additionally our approach assays the region upstream of the provirus in addition to the downstream region, giving additional information on the frequency of 5’ deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 1 BLV and HTLV-1 samples, representing both tumors and preleukemic stages. Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the 5’ end of the provirus identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting.

Phase I / II trial of dose adjusted EPOCH chemotherapy with bortezomib combined with integrase inhibitor therapy for HTLV-1 associated T-cell leukemia lymphoma

Adult T-cell leukemia lymphoma (ATLL) acute and lymphoma subtypes have a poor prognosis, with median survival of about one year. The malignant cells are characterized by high levels of nuclear factor kappa B (NFkappaB). In order to improve therapy, we assessed the safety, tolerance, and efficacy of a combination of dose-adjusted EPOCH chemotherapy (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin), combined with proteasome inhibitor, bortezomib, to prevent degradation of the inhibitor of NFkappaB (IkappaB). In addition, integrase inhibitor, raltegravir, was added to the regimen to block virus replication occurring during treatment. This multicenter study enrolled 18 of 20 planned subjects over 2.5 yrs in the U.S., although 15 of the subjects were born in the Caribbean. Six subjects had acute ATLL, and the remainder lymphoma subtype, all but one with stage 4 disease. Therapy was well tolerated; subjects received 1-6 cycles of therapy (mean 4.5 cycles). Two subjects achieved complete remission lasting for >12 mos, 10 subjects had a partial remission, and 3 subjects had stable disease as their best response. Baseline calcium level, absolute lymphocyte count, and proviral DNA load were not predictive of response. Correlations with proviral expression, integration site, and integrase gene analyses will be presented. Supported by NIH grants CA94056, CA1730, CA63413, Lymphoma Leukemia Society grant 6067-10, and Lymphoma Research Foundation grant 307181203. Authors’ details Division of Oncology, Washington University, St Louis, MO, USA. University of Miami, Miami, FL, USA. Lymphoma & Hematology Division, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Divisions of Hematology-Oncology, Montefiore Hospital, New York, NY, USA. Columbia University, New York, NY, USA. Division of Hematologic Malignancies, Johns Hopkins University, Baltimore, MD, USA. NINDS, NIH, Bethesda, MD, USA.

Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via de novo infection (infectious spread) and infected cell proliferation (mitotic spread), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). However, the relative contributions of these mechanisms are unknown, and will determine the efficacy of antiretroviral therapy. The prevailing view is that infectious spread does not contribute to HTLV-1 proviral load after early infection. However, this notion is based on low-throughput and imprecise data on clonal integration site abundance and diversity. We recently used our high-throughput protocol that maps and quantifies the abundance of HTLV-1 integration sites to estimate a diversity of between 10^4 and 10^5 HTLV-1 T cell clones in the body of an asymptomatic carrier or patient with HAM/TSP. Since infectious spread establishes new clones, high clonal diversity implies substantial infectious spread. HTLV-1 clonal abundance varies within individual hosts by several orders of magnitude. Therefore quantifying within-host HTLV-1 dynamics requires mathematical modelling at multiple scales, and stochastic processes are necessary to describe the behaviour of small clones. Here we apply a hybrid model of deterministic and stochastic processes to time-series patient data, to estimate the relative contributions of infectious spread and mitotic spread. We find, contrary to previous belief that infectious spread persists during chronic infection, even after the proviral load has reached its set point. The risk of HTLV-1-associated malignant and inflammatory disease is strongly correlated with the proviral load: the load in turn is correlated with the total number of HTLV-1-infected clones, but not with the degree of oligoclonal proliferation. Our results therefore suggest that attempts to suppress de novo infection may reduce the risk of malignant transformation.

T-cell receptor chain Vbeta subunit staining to quantify the malignant clone in adult T-cell leukemia

Adult T cell leukemia (ATL) is typically characterised by expansion of a single dominant malignantly transformed HTLV-1-infected clone. Clinically, the abundance of this clone can be estimated by flow cytometric assessment of CD4, CD25 and CD7 expression, and, more recently, by quantitative LM-PCR analysis of viral integration site. In addition to the unique proviral integration site, malignant clones arising from a single infection event also share an identical T cell receptor (TCR) sequence, utilising one of a potential 47 TCR Vbeta subunit genes. Using multiparametric flow cytometry, we quantified the frequency of peripheral T cells bearing each of 24 TCR Vbeta subunits in a cohort of individuals with adult T cell leukemia or lymphoma (n=26) at various stages of disease progression/remission. To establish an appropriate baseline, we also analysed T cells from asymptomatic carriers (n=11) and donors with HAM/TSP (n=13). Vbeta-specific antibodies stained 75% of CD3+ cells in donors without malignancy, and the median size of the largest single subset (CD4+Vbeta2+) was 7.6 % of CD3+ cells. We detected putative T cell clones which exceeded 20% of CD3+ T cells only in individuals with active chronic or acute leukemia (n=17), reaching frequencies of up to 96% of CD3+ T cells. With this method, we quantified the expression on clonally expanded populations of the putative ATL cell markers CD25, CCR4, TSLC1, CD7 and CD127. Almost 1% of expanded clonal populations in ATL patients were CCR4+TSLC1+CD7–. Surprisingly, in most donors only 60% of the dominant clonal population expressed high levels of CD25. This flow cytometric protocol is a potentially powerful method to both monitor disease progression and response to therapy, providing fast and accurate quantification of clonally expanded populations. We are currently employing this technique to assay lysis of expanded clones by autologous cytotoxic T lymphocytes in patients with ATL.

CD8 malignant proliferation in association with human T cell lymphotropic Virus 1 infection: a case report

Adult T cell leukaemia/lymphoma (ATL) is a predominantly CD4+ T cell neoplasia caused by human T cell lymphotropic Virus 1 (HTLV-1) infection. A few cases of CD8+ ATL have been reported but its existence has been debated. An Afrocaribbean female presented to the dermatology clinic with widespread papulo-nodular rash. HTLV-1 infection was detected by serology. Skin biopsy showed dense dermal infiltration by mainly CD8+ T cells which also expressed CD2, CD3, CD5, CD7 and occasionally CD25. Further investigations showed a mild lymphocytosis (4.4 x 106/L), circulating flower cells, absolute CD4+ count 1247 x 106/L (28.3% of lymphocytes), absolute CD8+ count x 2617 x 106/L (59.8% of lymphocytes), LDH 241 U/L (NR 135 – 214), Calcium 2.7 mmol/L (NR 2.2 – 2.6) but with increased serum PTH level. HTLV-1 proviral load (PVL) in peripheral blood mononuclear cells was 46%. PET/CT scan demonstrated FDG avid lymph nodes above and below the diaphragm. Axillary lymph node core biopsy showed CD20+ B cells nodules admixed with mainly small-to-medium sized T cells and a small population of large T cells. The small lymphocytes were mainly CD8+ T cells (also expressed CD2, CD3, CD5, and CD7 whereas the large cells expressed CD8, CD25 and CD30. Fewer than 5% expressed Ki67 (MIB-1). TCR clonality studies by PCR revealed expansion of an identical clone in all three compartments (skin, lymph node and blood). Detailed immunophenotyping on peripheral blood demonstrated a single dominant TCR vβ1+ clone (>90% of total CD8 T cells) which was CD3+, CD7+, CCR4+, CD25 low and, TSCL-1-. In summary we report a malignant CD8+ clone with atypical immunophenotype in a patient with high PVL. To confirm a working diagnosis of CD8+ ATL high throughput sequencing of each proviral integration sites is underway.

Abstract 5397: Characterization of INCB053914, a novel pan-PIM kinase inhibitor

Abstract The PIM (Proviral Integration sites for Moloney virus) family of constitutively active serine/threonine kinases consists of three members (PIM1, PIM2 and PIM3) that phosphorylate substrates involved in both apoptosis and cellular metabolism. As downstream targets of the JAK/STAT pathway and by having overlapping substrates with the PI3K/AKT pathway, PIM kinases integrate signals from multiple pathways important for the survival and proliferation of malignant cells. Overexpression of PIM kinases has been reported in human hematological cancers with each isoform showing a distinct expression pattern among the various malignancy subtypes, including MM, AML, ALL and NHL. Although the three family members possess overlapping functions that are mutually compensatory, mice deficient for all three PIM kinases exhibit minor phenotypic abnormalities, with smaller overall size at maturity and decreased cellular responses to cytokines. These data suggest that pan-inhibition of the PIM kinase family will have a favorable toxicity profile and may be required for therapeutic benefit in hematological malignancies. INCB053914 is a novel, ATP-competitive, small molecule, pan-inhibitor of PIM kinases. In biochemical assays, INCB053914 is potent against PIM1, PIM2 and PIM3 and highly selective. In cell proliferation assays, INCB053914 is active as a single agent in the majority of cell lines derived from different hematological malignancies, including MM, AML, DLBCL, MCL and T-ALL, with IC50 values ranging from 3 - 300 nM. INCB053914 synergizes with a variety of cytotoxic and targeted agents, reducing the viability of a panel of hematological tumor cell lines. In pharmacodynamic assays, INCB053914 inhibits phosphorylation of S6RP, P70S6K, 4E-BP-1 and BAD, known PIM kinase targets. Following treatment with INCB053914, induction of PIM kinase protein levels was noted in human cancer cell lines. Importantly, this compensatory response does not reduce the activity of the molecule, as effects on downstream signaling and efficacy remain intact when PIM kinase levels are elevated. Induction of PIM kinases after INCB53914 treatment was also evident in whole blood from healthy volunteers and in primary blast cells isolated from patients diagnosed with AML, further supporting the need for pan-PIM kinase inhibition. Taken together, these results indicate that INCB053914 has potent and selective activity against PIM kinases and may be useful as monotherapy or in combination with other agents for treating hematologic malignancies that are dependent on PIM kinase activity for growth and survival. Citation Format: Niu Shin, Maryanne Covington, Richard Wynn, Yanlong Li, Alexander Margulis, Qian Wang, Kathy Wang, Cindy Marando, Patricia Feldman, Lynn Leffet, Karen Gallagher, Xin He, Hong Chang, He Zhang, Hao Feng, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Timothy Burn, Kris Vaddi, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Gregory Hollis, Robert Newton, Reid Huber, Peggy Scherle, Holly Koblish. Characterization of INCB053914, a novel pan-PIM kinase inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5397. doi:10.1158/1538-7445.AM2015-5397

Abstract 3586: The use of kinome profiling to determine potential resistance pathways in pancreatic cancer cells treated with PIM kinase inhibitors

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent cancer of the pancreas with a 5-year survival rate of 6%, making it the 4th most common cause of cancer deaths in the United States. One of the challenges related to this cancer is the issue of chemoresistance. Chemoresistance renders most drugs ineffective in the treatment of PDAC, including kinase inhibitors and the chemotherapeutic drug gemcitabine. PIM (Proviral Integration site for the Moloney murine leukemia virus) kinases are a family of proteins shown to play an important role in the chemoresistance of many cancers, including pancreatic cancer. PIMs act downstream of the JAK-STAT cytokine signaling pathway and phosphorylate substrates involved in numerous cell functions including cell cycle progression and apoptosis. One objective of our research was to determine the role of PIM kinases on transformed phenotypes of PDAC cells. We were able to demonstrate that the PIM kinase inhibitor SGI-1776 and TP-3654, a second generation PIM kinase inhibitor, were able to inhibit growth in cell viability assay. Also, the PIM inhibitors were able to decrease various phenotypes such as anchorage-dependent and anchorage-independent growth in MIA PaCa-2 and PANC-1 cells. A second objective was to determine if PIM kinase inhibitors could reduce chemoresistance in PDAC cells. PIM inhibitors in combination with gemcitabine decreased cell viability in PDAC cells significantly more than when administered separately. To investigate resistance pathways activated in PDAC cells after treatment with PIM kinase inhibitors, we applied multiplexed inhibitor beads (MIBs) mass spectrometry (MS) to profile the kinome. We observed numerous kinases that were downregulated or upregulated after treatment. One of the kinases upregulated was IKKα, a key kinase in the anti-apoptotic NF-κB signaling pathway. Our results suggest that PIM kinases are an important target for cell growth and chemoresistance in pancreatic cancer. Also, these results suggest that treating PDAC cells with a PIM inhibitor simultaneously with an IKK inhibitor might prevent the effects of resistance pathways. Overall, our studies demonstrate that kinome profiling is an effective strategy to identify cellular responses to PIM kinase inhibitors as well as other drugs for treating pancreatic and other cancers. Citation Format: Vandana Singh, Brittany Nixon, T.S. Karim Gilbert, Steve Warner, David Bearss, Lee M. Graves, Antonio T. Baines. The use of kinome profiling to determine potential resistance pathways in pancreatic cancer cells treated with PIM kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2015-3586

Abstract 2671: INCB050465, a novel PI3Kδ inhibitor, synergizes with PIM protein kinase inhibition to cause tumor regression in a model of DLBCL

Abstract Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2), giving rise to phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in both B cell development and function, and has now been validated as a therapeutic target in CLL and indolent NHL upon the approval of idelalisib, a selective PI3Kδ inhibitor. However, despite the demonstrated activity of PI3Kδ inhibitors in several subtypes of B cell malignancies, the effectiveness of the approach as a single agent for the treatment of DLBCL, the largest subtype of NHL, has not been demonstrated. We hypothesized that an optimal treatment strategy in this histology would require combined inhibition of PI3Kδ with additional agents that target B cells through distinct signaling pathways. INCB050465 is a novel, small molecule inhibitor of PI3Kδ. In biochemical assays, it potently inhibits PI3Kδ (IC50 = 1 nM at 1 mM ATP) with approximately 20,000-fold selectivity for PI3Kα, PI3Kβ, PI3Kγ and 57 other kinases. In various PI3Kδ functional assays, including B cell proliferation induced by activation of BCR, BAFF-R, IL-4R, CD40 and TLRs as well as T cell differentiation assays, INCB050465 demonstrates potent activity with IC50 values ranging from 0.2 to 2 nM. In addition, INCB050465 blocks the proliferation of several DLBCL and MCL cell lines in vitro (EC50 &amp;lt; 10 nM) and can slow tumor growth in the Pfeiffer model of DLBCL in vivo. In cells, INCB050465 blocks constitutive PI3Kδ signaling, including phosphorylation of Akt and FOXO3a, with potencies similar to those for inhibiting proliferation. More importantly, the ability of INCB050465 to inhibit DLBCL cell proliferation in vitro can be further potentiated by inhibition of PIM (Proviral Integration sites for Moloney virus) protein kinases, which were also found to be important for B cell development and to phosphorylate substrates which overlap with those of the PI3K pathway. The combined inhibition of PI3Kδ by INCB050465 with the potent and selective PIM protein kinase inhibitor, INCB053914, results in profound tumor regression in the Pfeiffer cell model of DLBCL. Taken together, these results provide evidence for cross-talk between the PIM and PI3Kδ pathways, and support a mechanistic rationale for the clinical evaluation of combined PIM and PI3Kd inhibition in B cell malignancies, including DLBCL. Citation Format: Niu Shin, Holly Koblish, Maryanne Covington, Yanlong Li, Kathy Wang, Qian Wang, Patricia Feldman, Leslie Hall, Sybil O'Connor, Xin He, Kamna Katiyar, Yu Li, Eddy W. Yue, Thomas P. Maduskuie, Brent Douty, Song Mei, Yun-Long Li, Chu-Biao Xue, Andrew Combs, Wenqing Yao, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Robert Newton, Kris Vaddi, Reid Huber, Peggy Scherle. INCB050465, a novel PI3Kδ inhibitor, synergizes with PIM protein kinase inhibition to cause tumor regression in a model of DLBCL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2015-2671

Abstract 512: Pim kinases and Pim inhibitors in the regulation of prostate cancer cell migration and invasion

Abstract INTRODUCTION PIM kinases (Proviral Integration Site for Moloney Murine Leukemia Virus) are well known oncoproteins which have been shown to regulate cell survival, proliferation and motility. In addition, their expression is often up-regulated in human hematopoietic malignancies and solid tumors. Since PIM kinases promote cancerous behavior of cells, they have become attractive targets for drug development. We have previously identified a novel PIM-selective kinase inhibitor 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9). Here we show the potential of DHPCC-9 to efficiently prevent PIM-dependent functions both in cell-based assays and in mouse models. To elucidate signaling mechanisms via which PIM kinases regulate cell motility, we studied the involvement of the previously known PIM substrate NFATC1 (Nuclear Factor of Activated T Cells). Here we show that NFATC1 may play a role as a downstream mediator of PIM1 signaling in promoting cancer cell migration. METHODS PIM inactivation by DHPCC-9 or PIM silencing was studied in wound healing and Boyden chamber assays in PC-3 prostate cancer cells. The effects of stable PIM overexpression on PC-3 cell tumor growth and metastasizing were studied in subcutanic and in orthotopic mouse models. Mice bearing PC-3/PIM3 cell-derived orthotopic tumors were treated daily by injecting 50 mg/kg of DHPCC-9. Three weeks later tumors were measured, and all the substantial organs were collected for further analysis. Phosphorylation of NFATC1 was studied by mass spectrometry and in vitro kinase assays while its transcriptional activity was measured by luciferase reporter assays. In addition, wound healing assays were performed to test the effect of NFATC1 on PC-3 prostate cancer cell migration. RESULTS Prostate cancer cell migration and invasion were efficiently decreased by either PIM inhibition by DHPCC-9 or silencing of both PIM1 and PIM2 by RNA interference. Stable PIM1 or PIM3 overexpression promoted prostate tumor growth, while PIM inhibitor DHPCC-9 treatment decreased tumor size. Stable PIM overexpression induced PC-3 cells to metastasize into the prostate- draining lymph nodes and lungs, and treatment with PIM inhibitor DHPCC-9 decreased the amount of both types of metastases. Mutagenesis of all PIM1 phosphorylation target sites results in decreased NFATC1 phosphorylation and transcriptional activity, along with a reduced potential of NFATC1 to promote prostate cancer cell migration. CONCLUSIONS Here we have shown that PIM kinases promote and PIM inhibition reduces tumor growth and the formation of metastases in vivo. These findings not only support the role of PIM kinases in the regulation of cancer cell invasiveness, but also reveal DHPCC-9 as a highly potential lead compound for developing selective PIM-targeted cancer therapy. In addition, we have discovered NFATC1 transcription factor as the first PIM substrate involved in the PIM-promoted cancer cell motility. Citation Format: Sini Eerola, Niina Santio, Johanna Tuomela, Eeva-Marja Rainio, Pascale Moreau, Fabrice Anizon, Garry Corthals, Pirkko Härkonen, Päivi Koskinen. Pim kinases and Pim inhibitors in the regulation of prostate cancer cell migration and invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2015-512

230. A Novel Gammaretroviral Shuttle Vector Insertional Mutagenesis Screen To Identify Breast Cancer Metastasis Genes

Retroviral insertional mutagenesis screens have been used to efficiently identify genes involved in a wide variety of cancers. Identifying causal driver genes in malignant disease is crucial to develop improved targeted therapies. Here, we have utilized a replication-incompetent gammaretroviral vector to perform a mutagenesis screen to identify genes involved in breast cancer metastasis progression. MDA-MB-231 cells that were mutagenized with a gammaretroviral vector were xenotransplanted into the mammary fat pad of immunodeficient mice, and the primary tumors and metastases allowed to develop. Metastatic tumors were collected and analyzed for proviral integration sites using a shuttle vector rescue approach that allows for rapid rescue of plasmids in E. coli that contain long terminal repeat-chromosomal junctions. In our approach, clones with vector proviruses that dysregulate nearby metastatic driver genes have a selective advantage to form metastatic tumors. The vector provirus acts as a molecular tag to identify candidate dysregulated metastasis driver genes. Using this approach, we identified several candidate breast cancer metastasis genes including WWTR1 (TAZ), a known and recently validated breast cancer metastasis gene. Identifying genes that drive the metastatic process can identify targets for improved therapy.

Pim kinases significantly impact humoral immune responses (IRM12P.648)

Abstract The Provirus Integration site for Moloney murine leukemia virus (Pim) family of oncogenic serine/threonine kinases regulates a variety of metabolic functions in hematopoietic cells and lymphocytes. We examined the importance of Pim kinases in B cell activation following antigen stimulation. Humoral immune responses were significantly impaired in Pim 1 and 2 deficient mice (Pim 1-/-2-/-) T cell dependent responses were compromised while T cell independent type 1 and 2 antigen responses were virtually absent. Humoral deficiencies were owing to reduced production of antibody secreting cells due to the diminished induction of BLIMP-1 expression. The central role of Pim in these responses is consistent with our observation that Pims are induced downstream of multiple B cell activation receptors including CD40, BCR, and TLR4. We suggest that Pim 1 and 2 dependent signaling pathways are involved in B cell activation and their loss requires increased signaling through other pathways to reach an activation threshold. This hypothesis is further supported by the initial higher affinity of BCRs on Pim deficient B cells activated in a TD response and their inability to proliferate or undergo isotype switching in culture at the same rate as WT B cells when activator concentrations are decreased. Taken together, these data demonstrate a critical role for Pim kinases in affinity maturation, isotype switching, proliferation and the production of antibody secreting cells by B lymphocytes. .

Increased expression of activated pSTAT3 and PIM-1 in the pulmonary vasculature of experimental congenital diaphragmatic hernia

PurposeSignal transducer and activator of transcription (STAT) protein family (STAT1-6) regulates diverse cellular processes. Recently, the isoform STAT3 has been implicated to play a central role in the pathogenesis of pulmonary hypertension (PH). In human PH activated STAT3 (pSTAT3) was shown to directly trigger expression of the provirus integration site for Moloney murine leukemia virus (Pim-1), which promotes proliferation and resistance to apoptosis in SMCs. We designed this study to investigate the hypothesis that pSTAT3 and Pim-1 pulmonary vascular expression is increased in nitrofen-induced CDH. MethodsPregnant rats were exposed to nitrofen or vehicle on D9.5. Fetuses were sacrificed on D21 and divided into nitrofen (n=16) and control group (n=16). QRT-PCR, western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene and protein expression levels of pSTAT3 and Pim-1. ResultsPulmonary Pim-1 gene expression was significantly increased in the CDH group compared to controls. Western blotting and confocal-microscopy confirmed increased pulmonary protein expression of Pim-1 and increased activation of pSTAT3 in CDH lungs compared to controls. ConclusionMarkedly increased gene and protein expression of Pim-1 and activated pSTAT3 in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that pSTAT3 and Pim-1 are important mediators of PH in nitrofen-induced CDH.