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Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38−CD123+ and CD34+CD38−CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients’ outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37–0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment‐dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first‐line treatment. In primary CLL cells, inhibition of PIM kinases with a pan‐PIM inhibitor, SEL24‐B489, decreased PIM‐specific substrate phosphorylation and induced dose‐dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24‐B489 was similar in TP53‐mutant and TP53 wild‐type cells. Finally, inhibition of PIM kinases decreased CXCR4‐mediated cell chemotaxis in two related mechanisms‐by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4‐triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12‐triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment‐modulated PIM expression, their pro‐survival function and a role of PIMs in CXCR4‐induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in acute myeloid leukemia patients (AML). Although FLT3 tyrosine kinase inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. For such reasons, we have developed SEL24-B489 - a potent, dual PIM and FLT3-ITD inhibitor. SEL24-B489 exhibited significantly broader on-target activity in AML cell lines and primary AML blasts than selective FLT3-ITD or PIM inhibitors. SEL24-B489 also demonstrated marked activity in cells bearing FLT3 tyrosine kinase domain (TKD) mutations that lead to FLT3 inhibitor resistance. Moreover, SEL24-B489 inhibited the growth of a broad panel of AML cell lines in xenograft models with a clear pharmacodynamic-pharmacokinetic relationship. Taken together, our data highlight the unique dual activity of the SEL24-B489 that abrogates the activity of signaling circuits involved in proliferation, inhibition of apoptosis and protein translation/metabolism. These results underscore the therapeutic potential of the dual PIM/FLT3-ITD inhibitor for the treatment of AML.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets are functionally dependent on different survival mechanisms and exhibit distinct metabolic fingerprints. One of DLBCLs subtypes relies on mitochondrial oxidative phosphorylation and nutrient utilization pathways that provide pro-survival advantage independent of B-cell receptor (BCR) signaling. These OxPhos-DLBCLs are resistant to BCR inhibition and remain functionally poorly defined. We and others found that OxPhos-DLBCLs, compared to the BCR-dependent DLBCLs, overexpress heat shock protein HSP90alpha (Br J Haematol 2009; 144:358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. We found that HSP90alpha gene/protein expression correlated with SIRT1 protein level in DLBCL cell lines. SIRT1 knockdown or chemical inhibition reduced HSP90alpha expression in a mechanism involving HSF1, whereas HSP90 inhibitor (17AAG) reduced SIRT1 protein stability suggesting a chaperone function of HSP90alpha toward SIRT1 and a functional link between these proteins. We confirmed the SIRT1-HSP90alpha interaction in DLBCL cells using proximity ligation assay (PLA). The number of PLA complexes was significantly higher in OxPhos- (Ly4, K422, Toledo) than BCR- (Ly1, DHL4, Ly7, DHL6) DLBCL cell lines (p While low rates of chromosomal instability induces tumorigenesis, increased chromosomal missegregaton leads to cell death and suppresses cancer development. SIRT1 inhibitors (EX-527, cambinol, tenovin-6) induced dose-dependent cytotoxicity in DLBCL cell lines, while simultaneous addition of HSP90 inhibitor (17AAG) produced synergistic or additive effect in OxPhos-, but not BCR-DLBCLs. Taken together, our findings define a new OxPhos DLBCL-specific pathogenetic mechanism involving SIRT1 and HSP90alpha that regulates chromosome dynamics during mitosis and may be exploited therapeutically. Disclosures Juszczynski: Ryvu Therapeutics: Other: member of advisory board.

Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4, KLF4, CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo.

Diffuse large B-cell lymphomas (DLBCL) are heterogenous tumors. A large subset of DLBCL is reliant on B-cell receptor activity ('BCR'-DLBCLs). Additional subset of tumors that does not exhibit addiction to BCR signaling features transcriptional program is associated with increased oxidative phosphorylation ('OxPhos'-DLBCLs, Blood 2005; 105: 1851-61). These OxPhos tumros are resistant to BCR inhibition and remain functionally poorly defined. We and others found that OxPhos-DLBCLs, compared to BCR subtype, exhibit overexpression of heat shock protein HSP90alpha (Br J Haematol 2009; 144: 358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. However, molecular mechanisms responsible for increased HSP90alpha expression in OxPhos-DLBCLs remain unknown. We investigated the abundance of NAD-dependent deacetylase SIRT1 in a panel of DLBCL cell lines and found that SIRT1 protein level was higher in OxPhos- than BCR-DLBCLs and correlated positively with HSP90alpha gene expression (p=0.02, r=0.7857). To assess the role of SIRT1 in transcriptional regulation of HSP90alpha, we used a small molecule SIRT1 inhibitor tenovin-6 or interfering RNA, which dramatically reduced HSP90alpha induction after heat shock. We next characterized the mechanisms of SIRT1 overexpression in OxPhos cells. SIRT1 transcript abundance was similar in OxPhos and BCR cells, suggesting that changes in SIRT1 expression result from different protein half-life/stability. To test this assumption, OxPhos and BCR cells were incubated with a protein synthesis inhibitor, cycloheximide, and assessed for SIRT1 protein decays over time. Our experiments revealed increased stability of SIRT1 protein in OxPhos-DLBCLs, compared to BCR-DLBCLs. Addition of an HSP90 inhibitor 17-AAG accelerated SIRT1 protein degradation, suggesting that HSP90 might chaperone and thus protect SIRT1 against degradation. We further found that SIRT1 coimmunoprecipitated with HSP90alpha, confirming interaction between these proteins and highlighting a self-reinforcing feedback loop linking HSP90alpha and SIRT1 in OxPhos-DLBCLs. We next assessed the therapeutic potential of SIRT1 inhibition in DLBCLs. SIRT1 knockdown with small interfering RNA or chemical SIRT1 inhibition (tenovin-6) decreased proliferation of OxPhos-DLBCL cells, but also of BCR-DLBCLs, indicating that SIRT1 inhibition exhibits broader, subtype-independent activity in DLBCL cell lines. Surprisingly, BCR-type DLBCLs were significantly more sensitive to SIRT1 inhibitor, tenovin-6. We thus sought to identify a mechanism responsible for SIRT1 inhibition in BCR-DLBCLs. Since SIRT1 deacetylase modulates the transcriptional activity of BCL6 oncogene, critical for survival of BCR-type DLBCLs (PNAS 2007; 104: 3207-12), we assessed the expression of BCL6-regulated genes in these cells. SIRT1 inhibition upregulated the abundance of multiple BCL6-dependent transcripts (including FCER2, CCL3, BLIMP1 and CD74), suggesting that toxicity of SIRT1 inhibition in BCL6-dependent DLBCLs is, at least partially, mediated by decreased BCL6 repressor activity. Although OxPhos-DLBCLs were less sensitive to tenovin-6 than BCR-type DLBCLs, we hypothesized that, given the feedback loop between SIRT1 and HSP90, simultaneous inhibition of HSP90 would increase the cellular sensitivity to tenovin-6. Consistent with this, we found that combination of these two compounds exhibited synergistic activity in OxPhos-DLBCL. Taken together, our data demonstrate that SIRT1 and HSP90alpha are mutually linked and involved in the pathogenesis of OxPhos-DLBCL. Targeting SIRT1-HSP90alpha loop with combinatorial use of SIRT1 and HSP90 inhibitors might be a promising treatment strategy in OxPhos-DLBCLs. Disclosures Warzocha: BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells. We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis 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, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform. We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors. Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells. PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway. Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy. Disclosures Warzocha: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.