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.

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.

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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.

Background: Use of targeted therapy for B-cell malignancies has resulted in significant improvements in patient overall-and progression-free-survival. These treatments have focused on inhibition of components of the B-cell receptor (BCR) such as BTK and PI3Kd, targeted by ibrutinib and idelalisib respectively, and are often used in combination with other agents such as Microtubule Targeting Agents (MTA), alkylating agents and glucocorticoids. However, relapse and resistant disease remain a significant challenge. Thienopyridines, commonly used as anti-thrombotic therapy, have recently been identified as potential anti-cancer agents, with PLCg2 (acting downstream of BTK in BCR signalling), Jak2 and Lck suggested as direct targets, while mitotic spindle inhibition via microtubule de-stabilisation has also been proposed. Theinopyridines may therefore have potential as a future treatment for B-cell malignancies. Aims: This study aimed to investigate the anti-proliferative and microtubule-disrupting properties of ten novel thienopyridine compounds synthesised by our group, in B-cell Lymphoma cells in-vitro, to determine whether this class of compound has potential as future targeted therapy for B-cell malignancies. Methods: DAUDI Burkit Lymphoma cells were cultured in the presence of novel thienopyridines for 24-72 h at a range of concentrations (100 µM to 1 nM). Synthesis of the thienopyridine compounds (DJ0014, DJ0021, DJ0041, DJ0081, DJ0109, DJ0171, DJ0199, DJ0206 and DJ0209[ND1] ) has been previously published. Cellular biochemical activity was assessed using the MTS assay, while apoptosis and necrosis were observed using the Annexin V/Propidium Iodide (AV/PI) flow cytometry assay. Cell cycle arrest was determined by flow cytometric Propidium Iodide cell cycle assay, and alpha-tubulin expression was visualised by confocal microscopy following staining with α-Tubulin (DM1A) Mouse mAb (Alexa Fluor ® 488 Conjugate) antibody and DAPI nucleic acid stain. The rate of tubulin polymerisation in the presence of the compounds was assessed by Tubulin Polymerisation Assay, with the rate of polymerisation measured using spectrophotometry. Paclitaxel was used as a positive control for tubulin stabilisation.Results: Across all assays at 48 and 72 h, compounds DJ0014, DJ0041, DJ0081, DJ0199 and DJ0206 resulted in significantly reduced cell viability by MTS assay and increased early and late apoptosis by AV/PI, with corresponding reduction in live cell populations and absence of necrosis at 10 µM and 100 µM concentrations. Increased tetraploidy / G 2 /M arrest was also seen following treatment with the same compounds at 10 µM and 100 µM concentrations. These compounds showed a significant inhibitory effect on tubulin polymerisation at 10 µM dosage and signs of microtubule disruption via confocal microscopy. Summary/Conclusion:The study demonstrates the anti-mitotic activity of these novel thienopyridine derivatives in malignant B-cells and their potential for use in the treatment of mature B-cell malignancies. Future work aims to c...