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G protein coupled receptors (GPCRs) have emerged as the most potential target for a number of drug discovery programs ranging from control of blood pressure, diabetes, cure for genetic diseases to treatment of cancer. A panel of different ligands including hormones, peptides, ions and small molecules is responsible for activation of these receptors. Molecular genetics has identified key GPCRs, whose mutations or altered expressions are linked with tumorgenicity. In this review, we discussed recent advances regarding the involvement of GPCRs in the development of cancers and approaches to manipulating the mechanism behind GPCRs involved tumor growth and metastasis to treat different types of human cancer. This review provides an insight into the current scenario of GPCR-targeted therapy, progress to date and the challenges in the development of anticancer drugs., Graphical abstract GPCRs based drugs against different cancers.Image 1, Highlights • An association of GPCRs in tumor growth and metastasis is confirmed in different types of cancers. • Despite the incredible potential of GPCRs in cancer therapy, only a few anti-GPCR drugs has been approved for clinical use. • Advancement in studying the association of cancer with GPCRs is inevitable to manipulate these receptors for cancer therapy.

Chronic lymphocytic leukemia (CLL) is still an incurable disease despite aggressive chemotherapies including the B-cell receptor (BCR) targeted-inhibitors. Therefore, we assessed the expression status of key signal mediators of the BCR pathway in CLL cells. Indeed, we detected aberrantly elevated levels of CD79a, B-cell adaptor for PI3K (BCAP) and phospholipase C (PLC)γ2, key mediators of BCR signal, in CLL cells. As HSP90 is also overexpressed in CLL cells, we hypothesized that HSP90 could potentiate the BCR signal via stabilization of multiple key components of the BCR-signalosome. We found that HSP90 formed a multi-molecular complex with CD79a, BCAP, PLCγ2, LYN, SYK, Bruton tyrosine kinase (BTK) and AKT and that, pharmacologic inhibition or partial depletion of HSP90 reduced the expression of these signal mediators in CLL cells. In addition, our findings also demonstrated that HSP90 could stabilize the tyrosine phosphatase, PTPN22 which positively regulates AKT phosphorylation, and the constitutively active fibroblast growth factor receptor 3 (FGFR3) in CLL cells. Finally, HSP90 inhibition induced apoptosis in CLL cells in a dose-dependent manner likely via downregulation of anti-apoptotic proteins MCL-1 and XIAP, but not BCL2, reported to be overexpressed in CLL cells. In total, our findings suggest that HSP90-inhibition may sensitize the leukemic B-cells to BCR-targeted agents, particularly those become resistant to these therapies.

This chapter discusses the work of the Futurist poet and broadcaster Luciano Folgore in the light of his emphasis on the mechanical body of Pinocchio. Spanning Folgore’s long and eclectic career, this emphasis is seen in the wider context of a Futurist advocacy of technology and machines in the early years of the twentieth century. Now archived in the John Paul Getty Research Institute and Library in Los Angeles, Folgore’s copious production reveals a serious engagement with the mechanic nature of the puppet, as well as a veritable fixation with Pinocchio’s robotic nose. The puppet’s artificial nose becomes increasingly a marker of his mechanical identity.

Circular RNAs (circRNAs) have recently been reported to play crucial roles in various regulatory processes and involved in cancer onset and progression. However, the potential mechanism of circRNAs in chronic lymphocytic leukemia (CLL) remains largely unknown. Here, we observed hsa_circ_0132266 (circ_0132266), a circRNA significantly decreased in the peripheral blood mononuclear cells (PBMCs) of CLL patients compared with healthy donors, could act as an endogenous sponge of hsa-miR-337-3p (miR-337-3p) and regulate its activity, which resulted in a downstream change of target-gene PML and a consequent influence on cell viability. Taken together, our data indicated the regulatory mechanism of circ_0132266 in CLL progression through circ_0132266/miR-337-3p/PML axis, suggesting that it may serve as a biomarker as well as an exploitable therapeutic target for CLL.

The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1 activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1 and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1 as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.

Summary Bendamustine + rituximab (BR) is the current first‐line standard‐of‐care for chronic lymphocytic leukaemia (CLL) in fit patients aged 66–70 years, whereas chlorambucil + CD20 antibody is recommended in older patients with co‐morbidities. This retrospective real‐world study investigated whether risk‐adapted BR was safe and effective in elderly patients. All 141 CLL patients in the Stockholm region (diagnosed from 2007 to 2016, identified from regional registries) who had received BR as first (n = 84) or later line (n = 57) were analysed. Median age was 72 years, 49% had Binet stage C, 40% had Cumulative Illness Rating Scale (CIRS) score ≥ 6, 20% Eastern Cooperative Oncology Group (ECOG) score 2. None had del(17p). Only 15% of patients aged ≥80 years received full‐dose bendamustine and 65% of them postponed rituximab until cycle 2. Corresponding numbers in patients 73–79 years were 21% and 36% and in

Earlier we have shown the expression of a constitutively active receptor tyrosine kinase Axl in CLL B-cells from previously untreated CLL patients, and that Axl inhibitor TP-0903 induces robust leukemic B-cell death. To explore whether Axl is an effective target in relapsed/refractory CLL patients, we analyzed CLL B-cells obtained from CLL patients on ibrutinib therapy. Ibrutinib-exposed CLL B-cells were treated with increasing doses (0.01- 0.50μM) of a new formulation of high-affinity Axl inhibitor, TP-0903 (tartrate salt), for 24 hours and LD50 doses were determined. Sensitivity of CLL B-cells was compared with known prognostic factors and effect of TP-0903 was also evaluated on Axl signaling pathway in CLL B-cells from this cohort. We detected sustained overexpression of Axl in CLL B-cells from CLL patients on ibrutinib treatment, suggests targeting Axl could be a promising strategy to overcome drug resistance and killing of CLL B-cells in these patients. We found that CLL B-cells from sixty-nine percent of relapsed CLL patients actively on ibrutinib therapy were found to be highly sensitive to TP-0903 with induction of apoptosis at nanomolar doses (≤0.50 μM). TP-0903 treatment effectively inhibited Axl phosphorylation and reduced expression levels of anti-apoptotic proteins (Mcl-1, XIAP) in ibrutinib exposed CLL B-cells. In total, our in vitro preclinical studies showing that TP-0903 is very effective at inducing apoptosis in CLL B-cells obtained from ibrutinib-exposed patients supports further testing of this drug in relapsed/refractory CLL.

Although several studies established that unlike normal B cells chronic lymphocytic leukemia (CLL) cells metabolize fatty acids (FA), how CLL cells internalize FA is poorly understood. Because in various cell types CD36 facilitates FA uptake, we wondered whether a similar mechanism is operative CLL. We found that CD36 levels are higher in CLL cells than in normal B cells, and that small interfering RNA, CD36 neutralizing antibodies or sulfosuccinimidyl oleate (SSO) that inhibits CD36 significantly reduced the oxygen consumption of CLL cells incubated with FA. Because CD36 is oeverexpressed and STAT3 is constitutively activated in CLL cells, we wondered whether STAT3 induces CD36 expression. Sequence analysis identified putative STAT3 binding sites in the CD36 gene promoter. Chromatin immunoprecipitation and an electrophoretic mobility shift assay revealed that STAT3 binds to the CD36 gene promoter. A luciferase assay and STAT3-small hairpin RNA, that significantly decreased the levels of CD36 in CLL cells, established that STAT3 activates the transcription of the CD36 gene. Furthermore, SSO induced a dose-dependent apoptosis of CLL cells. Taken together, our data suggest that STAT3 activates CD36 and that CD36 facilitates FA uptake in CLL cells. Whether CD36 inhibition would provide clinical benefits in CLL remains to be determined.

The functional significance of AKT in chronic lymphocytic leukemia (CLL) remains unclear. Given the importance of non-malignant T cells in regulating clonal expansion in CLL, we investigated the role of AKT in T cell-mediated cytoprotection and proliferation using an established co-culture system in which primary CLL cells were incubated on a monolayer of transfected mouse fibroblasts expressing human CD40L (CD154). Stimulation of CLL cells via CD40 induced activation of AKT, which was closely associated with downregulation of its negative regulator PTEN, and protected CLL cells from killing by bendamustine. This cytoprotective effect of CD40 stimulation was prevented by a selective inhibitor of AKT. Stimulation of CLL cells with CD154 + IL-4 or IL-21 induced proliferation detected as reduced fluorescence of cells pre-stained with CFSE. AKT inhibition produced a significant, consistent reduction in proliferation induced by CD154 + IL-4 and a reduction in proliferation induced by CD154 + IL-21 in most but not all cases. In contrast, AKT inhibition had no effect on the proliferation of normal B cells induced by CD154 + IL-4 or IL-21. These findings indicate that AKT contributes in a significant way to T-cell mediated survival and proliferation signalling in CLL and support the clinical evaluation of AKT inhibitors in this disease.

ATM, primarily activated by DNA double-strand breaks, and ATR, activated by single-stranded DNA, are master regulators of the cellular response to DNA damage. In primary chronic lymphocytic leukemia (CLL) cells, ATR signaling is considered to be switched off due to ATR downregulation. Here, we hypothesized that ATR, though expressed at low protein level, could play a role in primary resting CLL cells after genotoxic stress. By investigating the response of CLL cells to UV-C irradiation, a prototypical activator of ATR, we could detect phosphorylation of ATR at Thr-1989, a marker for ATR activation, and also observed that selective ATR inhibitors markedly decreased UV-C-induced phosphorylation of ATR targets, including H2AX and p53. Similar results were obtained with the purine analogs fludarabine and cladribine that were also shown to activate ATR and induce ATR-dependent phosphorylation of H2AX and p53. In addition, ATR inhibition was found to sensitize primary CLL cells to UV-C by decreasing DNA repair synthesis. Conversely, ATR inhibition rescued CLL cells against purine analogs by reducing expression of the pro-apoptotic genes PUMA and BAX. Collectively, our study indicates that ATR signaling can be activated in resting CLL cells and play a pro-survival or pro-apoptotic role, depending on the genotoxic context.

We investigated whether karyotype analysis and mutational screening by next generation sequencing could predict outcome in 101 newly diagnosed chronic lymphocytic leukemia patients with high-risk features, as defined by the presence of unmutated IGHV gene and/or 11q22/17p13 deletion by FISH and/or TP53 mutations. Cytogenetic analysis showed favorable findings (normal karyotype and isolated 13q14 deletion) in 30 patients, unfavorable (complex karyotype and/or 17p13/11q22 deletion) in 34 cases and intermediate (all other abnormalities) in 36 cases. A complex karyotype was present in 21 patients. Mutations were detected in 56 cases and were associated with unmutated IGHV status (p = 0.040) and complex karyotype (p = 0.047). TP53 disruption (i.e. TP53 mutations and/or 17p13 deletion by FISH) correlated with the presence of ≥ 2 mutations (p = 0.001) and a complex karyotype (p = 0.012). By multivariate analysis, an advanced Binet stage (p < 0.001) and an unfavorable karyotype (p = 0.001) predicted a shorter time to first treatment. TP53 disruption (p = 0.019) and the unfavorable karyotype (p = 0.028) predicted a worse overall survival. A shorter time to chemorefractoriness was associated with TP53 disruption (p = 0.001) and unfavorable karyotype (p = 0.025). Patients with both unfavorable karyotype and TP53 disruption presented a dismal outcome (median overall survival and time to chemorefractoriness of 28.7 and 15.0 months, respectively). In conclusion, karyotype analysis refines risk stratification in high-risk CLL patients and could identify a subset of patients with highly unfavorable outcome requiring alternative treatments.

GDC-0853 is a selective, reversible, and non-covalent inhibitor of Bruton’s tyrosine kinase (BTK) that does not require interaction with the Cys481 residue for activity. In this first-in-human phase 1 study we evaluated safety, tolerability, pharmacokinetics, and activity of GDC-0853 in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL). Twenty-four patients, enrolled into 3 cohorts, including 6 patients who were positive for the C481S mutation, received GDC-0853 at 100, 200, or 400 mg once daily, orally. There were no dose limiting toxicities. GDC-0853 was well tolerated and the maximum tolerated dose (MTD) was not reached due to premature study closure. Common adverse events (AEs) in ≥ 15% of patients regardless of causality included fatigue (37%), nausea (33%), diarrhea (29%), thrombocytopenia (25%), headache (20%), and abdominal pain, cough, and dizziness (16%, each). Nine serious AEs were reported in 5 patients of whom 2 had fatal outcomes (confirmed H1N1 influenza and influenza pneumonia). A third death was due to progressive disease. Eight of 24 patients responded to GDC-0853: 1 complete response, 4 partial responses, and 3 partial responses with lymphocytosis, including 1 patient with the C481S mutation. Two additional C481S mutation patients had a decrease in size of target tumors (–23% and –44%). These data demonstrate GDC-0853 was generally well-tolerated with antitumor activity.

// Adam Albitar 1 , Wanlong Ma 1 , Ivan DeDios 1 , Jeffrey Estella 1 , Inhye Ahn 2 , Mohammed Farooqui 3 , Adrian Wiestner 3 , Maher Albitar 1 1 NeoGenomics Laboratories, Irvine, CA, USA 2 Medical Oncology Service, National Cancer Institute, Bethesda, MD, USA 3 Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD, USA Correspondence to: Maher Albitar, email: malbitar@neogenomics.com Keywords: ibrutinib, CLL, resistance, PLCγ2, BTK Received: August 23, 2016 Accepted: January 27, 2017 Published: February 13, 2017 ABSTRACT Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naive patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naive CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

// Prexy Modi 1 , Kumudha Balakrishnan 1 , Qingshan Yang 1 , William G. Wierda 2 , Michael J. Keating 2 and Varsha Gandhi 1,2 1 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Varsha Gandhi, email: // Keywords : CLL, bendamustine, idelalisib, DNA damage, B cell receptor Received : January 16, 2017 Accepted : January 25, 2017 Published : February 07, 2017 Abstract Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.

// Cody Paiva 1, * , J. Claire Godbersen 2, * , Taylor Rowland 1 , Olga V. Danilova 3 , Christopher Danes 4 , Allison Berger 4 , Alexey V. Danilov 1 1 Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA 2 Geisel School of Medicine at Dartmouth, Department of Medicine, Hanover, NH, USA 3 VA Portland Healthcare System, Department of Pathology and Laboratory Medicine, Portland, OR, USA 4 Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd., Cambridge, MA, USA * These authors have contributed equally to this work Correspondence to: Alexey V. Danilov, email: danilov@ohsu.edu Keywords: lymphoma, neddylation, CLL, TRAIL Received: September 13, 2016 Accepted: January 07, 2017 Published: February 03, 2017 ABSTRACT While death receptor ligands (Fas and TRAIL) kill chemoresistant tumor cell lines, related therapies have limited clinical efficacy as single agents. Death receptor signaling is modulated by nuclear factor-κB (NFκB), a family of transcription factors which are constitutively active in B-cell malignancies. We and others have shown that pevonedistat, an investigational inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in B-cell neoplasia. Here we demonstrate that diffuse large B-cell lymphoma, particularly activated B-cell type, and primary chronic lymphocytic leukemia cells are re-sensitized to extrinsic apoptosis by pevonedistat. Pevonedistat enhanced caspase-8 processing following death receptor ligation, and downmodulated cFLIP, a NFκB-regulated protease-deficient caspase homolog. However, treatment with pevonedistat did not modulate death-inducing signaling complex in neoplastic B-cells, suggesting that they were sensitized to death ligands through the mitochondrial pathway. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies.

Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.

// Ailin Guo 1,* , Pin Lu 1,* , Greg Coffey 2 , Pamela Conley 2 , Anjali Pandey 2 and Y. Lynn Wang 1 1 Department of Pathology, Lymphoma Translational Pathology, University of Chicago, Chicago, IL, USA 2 Portola Pharmaceuticals, Inc., South San Francisco, CA, USA * These authors have contributed equally to this study Correspondence to: Y. Lynn Wang, email: // Keywords : CLL, cerdulatinib, SYK, JAK-STAT, molecularly targeted therapy Received : December 21, 2016 Accepted : January 01, 2017 Published : January 10, 2017 Abstract Ibrutinib (BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Notably, cerdulatinib blocked proliferation of ibrutinib-resistant primary CLL cells and of BTK C481S -transfected/ibrutinib-resistant lymphoma cells. These anti-tumor effects are well correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition.

Summary Otlertuzumab (TRU-016) is a humanized anti-CD37 protein therapeutic that triggers direct caspase-independent apoptosis of malignant B cells and induces antibody-dependent cell-mediated cytotoxicity. Patients with relapsed chronic lymphocytic leukaemia (CLL) received either otlertuzumab (20 mg/kg) weekly by IV infusion for two 28-day cycles then every 14 days for four 28-day cycles and IV bendamustine (70 mg/m2) on Days 1 and 2 of each cycle for up to six 28-day cycles or bendamustine alone. Thirty-two patients were treated with otlertuzumab and bendamustine and 33 with bendamustine alone. Overall response rate according to the International Workshop on Chronic Lymphocytic Leukaemia criteria was 69% in the otlertuzumab and bendamustine arm and 39% in the bendamustine alone arm (P = 0·025). Median progression-free survival (PFS) was 15·9 months in the otlertuzumab and bendamustine arm and 10·2 months in the bendamustine alone arm (P = 0·0192). There was a higher incidence of pyrexia (34% vs. 12%) and neutropenia (59% vs. 39%) with the combination but this did not result in a higher incidence of severe (grade 3/4) infections (13% vs. 27%). This combination significantly increased the response rate and prolonged the PFS over single agent bendamustine in patients with relapsed or refractory CLL.

// Neil E. Kay 1 , Paolo Strati 1 , Betsy R. LaPlant 1 , Jose F. Leis 2 , Daniel Nikcevich 3 , Timothy G. Call 1 , Adam M. Pettinger 1 , Connie E. Lesnick 1 , Curtis A. Hanson 1 and Tait D. Shanafelt 1 1 Mayo Clinic College of Medicine, Rochester, MN, USA 2 Mayo Clinic College of Medicine, Scottsdale, AZ, USA 3 Essentia Health’s East Region, Duluth, MN, USA Correspondence to: Neil E. Kay, email: // Keywords : CLL, chemoimmunotherapy, bevacizumab Received : October 15, 2016 Accepted : November 09, 2016 Published : November 16, 2016 Abstract Bevacizumab is a monoclonal antibody targeting vascular endothelial growth factor (VEGF) with in vitro pro-apoptotic and antiangiogenic effects on chronic lymphocytic leukemia (CLL) cells. As monotherapy in patients with CLL, it has no clinical activity. Here we report the results of an open-label, randomized phase II trial comparing the combination of pentostatin, cyclophosphamide and rituximab (PCR) either without or with bevacizumab (PCR-B) in previously untreated CLL patients. A total of 65 evaluable patients were enrolled, 32 receiving PCR and 33 PCR-B. A higher rate of grade 3-4 cardiovascular toxicity was observed with PCR-B (33% vs . 3%, p < 0.003). Patients treated with PCR-B had a trend for a higher complete remission (CR) rate (54.5% vs 31.3%; p = 0.08), longer progression-free survival (PFS)( p = 0.06) and treatment-free survival (TFS)( p = 0.09). No differences in PFS and TFS by IGHV mutational status were observed with the addition of bevacizumab. A significant post-treatment increase in VEGF levels was observed in the PCR-B arm (29.77 to 57.05 pg/mL); in the PCR-B arm, lower baseline CCL-3 levels were significantly associated with achievement of CR ( p = 0.01). In conclusion, the addition of bevacizumab to chemoimmunotherapy in CLL is generally well-tolerated and appears to prolong PFS and TFS.

19 p.-9 fig.-1 tab., CLL remains an incurable disease in spite of the many new compounds being tested. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the gene expression profile induced by ATO in CLL cells. ATO modulated many genes, largely involved in oxidative stress, being HMOX1 the most upregulated gene, also induced at the protein level. ATO also increased MMP-9, as we previously observed, both at the mRNA and protein level. Using specific inhibitors, qPCR analyses, and gene silencing approaches we demonstrate that upregulation of MMP-9 by ATO involved activation of the p38 MAPK/AP-1 signaling pathway. Moreover, gene silencing HMOX1 or inhibiting HMOX1 activity enhanced p38 MAPK phosphorylation and c-jun expression/activation, resulting in transcriptional upregulation of MMP-9. Overexpression of HMOX1 or enhancement of its activity, had the opposite effect. Cell viability analyses upon modulation of HMOX1 expression or activity demonstrated that HMOX1 had a proapoptotic role and enhanced the cytotoxic effect of ATO in CLL cells. We have therefore identified a new mechanism in which HMOX1 plays a central role in the response of CLL cells to ATO and in the regulation of the anti-apoptotic protein MMP-9. Thus, HMOX1 arises as a new therapeutic target in CLL and the combination of HMOX1 modulators with ATO may constitute an efficient therapeutic strategy in CLL., This work was supported by grants SAF2012-31613 and SAF2015-69180R (AGP) and RTICC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0061 (AGP), from the Ministry of Economy and Competitiveness (MINECO), Spain; S2010/BMD-2314-Neoplasbim (AGP) from the Comunidad de Madrid/European Union; and a grant from the Fundación para la Investigación Biomédica Hospital Universitario Puerta de Hierro, Madrid (JAGM).

// Nadja Zaborsky 1, 2 , Franz Josef Gassner 1, 2 , Daniela Asslaber 1, 2 , Petra Reinthaler 1, 2 , Ursula Denk 1, 2 , Sabine Flenady 1, 2 , Josefina Pinon Hofbauer 1, 2 , Barbara Danner 1, 2 , Stefan Rebhandl 1, 2 , Andrea Harrer 3 , Roland Geisberger 1, 2 , Richard Greil 1, 2 , Alexander Egle 1, 2 1 Laboratory for Immunological and Molecular Cancer Research, Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology and Rheumatology, Oncologic Center, Paracelsus Medical University, Salzburg, Austria 2 Salzburg Cancer Research Institute, Salzburg, Austria 3 Department of Neurology, Paracelsus Medical University, Salzburg, Austria Correspondence to: Nadja Zaborsky, email: n.zaborsky@salk.at Keywords: CLL, T cells, T cell skewing, CD1d, CD161 Received: August 25, 2015 Accepted: June 16, 2016 Published: July 01, 2016 ABSTRACT Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7 + NK1.1 + T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3 + cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161 + cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161 + T cells that may represent a novel therapeutic target for immune modulation.

// Patricia A. Toniolo 1, 2 , Suhu Liu 1 , Jennifer E. Yeh 1 , Darwin Q. Ye 1 , Jose Alexandre M. Barbuto 2 , David A. Frank 1 1 Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 2 Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil Correspondence to: David A. Frank, email: david_frank@dfci.harvard.edu Keywords: monocytes, dendritic cells, SOCS5, CLL, STAT3 Received: December 01, 2015 Accepted: May 28, 2016 Published: June 15, 2016 ABSTRACT One cause of morbidity and mortality in chronic lymphocytic leukemia (CLL) is infection, which results from defects in a number of components of the immune system. In particular, dendritic cells (DCs) are functionally defective in patients with CLL. To understand the molecular mechanism for this abnormality, we focused on signal transduction pathways that regulate the function of monocyte-derived dendritic cells (Mo-DCs). Monocytes from CLL patients exhibit high IL-4Rα expression due to the enhanced activation of STAT3. However, IL-4R signaling is decoupled from activation of its downstream mediator STAT6 by enhanced levels of the negative regulator SOCS5. This impairs differentiation of functionally mature DCs leading to decreased expression of HLA-DR and costimulatory molecules, and reduced secretion of pro-inflammatory cytokines in LPS-activated DCs. Moreover, Mo-DCs from CLL patients display a decreased ability to induce pro-inflammatory T-cell responses. IL-10-treatment of monocytes from healthy donors mimics the alteration in signaling observed in CLL patients, through enhanced STAT3-dependent expression of SOCS5. The higher level of SOCS5 inhibits STAT6 activation and leads to defective DC differentiation. These findings indicate that SOCS5 mediates the impaired function of DCs in CLL patients, and has the potential to be a new therapeutic target for reversing cancer-associated immune suppression.

// Andrea Tomasella 1 , Raffaella Picco 1 , Sonia Ciotti 1 , Andrea Sgorbissa 1 , Elisa Bianchi 2 , Rossella Manfredini 2 , Fabio Benedetti 3 , Valentina Trimarco 4 , Federica Frezzato 4 , Livio Trentin 4 , Gianpietro Semenzato 4 , Domenico Delia 5 , Claudio Brancolini 1, 2 1 Department of Medical and Biological Sciences, Universita degli Studi di Udine, Udine, Italy 2 Centre for Regenerative Medicine “Stefano Ferrari”, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 3 Dipartimento di Scienze Chimiche e Farmaceutiche, Universita degli Studi di Trieste, Trieste, Italy 4 Department of Medicine, Hematology and Clinical Immunology Branch, Padua University School of Medicine, Padua, Italy 5 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Correspondence to: Claudio Brancolini, email: claudio.brancolini@uniud.it Keywords: CLL, apoptosis, proteasome, proteotoxic stress, deubuquitylases Received: November 15, 2015 Accepted: May 22, 2016 Published: May 31, 2016 ABSTRACT Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.

// Vipul Shukla 1 , Ashima Shukla 1 , Shantaram S. Joshi 1 and Runqing Lu 1,* 1 Department of Genetics Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA * Has deceased Correspondence to: Vipul Shukla, email: // Keywords : IRF4, Notch signaling, CLL, B1 cells Received : May 03, 2016 Accepted : May 14, 2016 Published : May 25, 2016 Abstract Molecular pathogenesis of Chronic Lymphocytic Leukemia (CLL) is not fully elucidated. Genome wide association studies have linked Interferon Regulatory Factor 4 (IRF4) to the development of CLL. We recently established a causal relationship between low levels of IRF4 and development of CLL. However, the molecular mechanism through which IRF4 suppresses CLL development remains unclear. Deregulation of Notch signaling pathway has been identified as one of the most recurrent molecular anomalies in the pathogenesis of CLL. Yet, the role of Notch signaling as well as its regulation during CLL development remains poorly understood. Previously, we demonstrated that IRF4 deficient mice expressing immunoglobulin heavy chain Vh11 (IRF4 -/- Vh11) developed spontaneous CLL with complete penetrance. In this study, we show that elevated Notch2 expression and the resulting hyperactivation of Notch signaling are common features of IRF4 -/- Vh11 CLL cells. Our studies further reveal that Notch signaling is indispensable for CLL development in the IRF4 -/- Vh11 mice. Moreover, we identify E3 ubiquitin ligase Nedd4, which targets Notch for degradation, as a direct target of IRF4 in CLL cells and their precursors. Collectively, our studies provide the first in vivo evidence for an essential role of Notch signaling in the development of CLL and establish IRF4 as a critical regulator of Notch signaling during CLL development.

// Monika Podhorecka 1 , Dorota Halicka 2 , Agnieszka Szymczyk 1 , Arkadiusz Macheta 1 , Sylwia Chocholska 1 , Marek Hus 1 , Zbigniew Darzynkiewicz 2 1 Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland 2 Department of Pathology, New York Medical College, Valhalla, New York, USA Correspondence to: Monika Podhorecka, email: monika.podhorecka@onet.pl Keywords: CLL, RDW, mTOR, ZAP-70, CD38 Received: February 09, 2016 Accepted: April 08, 2016 Published: April 27, 2016 ABSTRACT Red blood cell distribution width (RDW) is a quantitative measure of the variability in size of circulating erythrocytes. It was recently reported that RDW is a prognostic factor for infection diseases, cardiovascular and pulmonary diseases, as well as some neoplasms. Moreover, RDW is remarkably strong predictor of longevity, including all causes of death, for adults aged 45 years and older. To explain this occurrence it was proposed that persistent IGFs/mTOR signaling is one of the factors that play a role in affecting the RDW and mortality. The above observations induced us to analyze the prognostic role of RDW in chronic lymphocytic leukemia (CLL) being the most frequent type of adult leukemia in Western countries. The obtained results have shown that RDW may be considered as a potential CLL prognostic marker. Elevated RDW level at the moment of diagnosis was associated with advanced disease and presence of other poor prognostic factors. It is also connected with overall survival indicating shorter time in patients with elevated RDW. It is possible that the presently observed correlation between mortality and RDW of the CLL patients is affected by their metabolic (IGF-1/mTOR driven)- rather than chronological- aging. The patients with high level of RDW are expected to have an increased persistent level of IGF-1/mTOR signaling. Within the framework of personalized therapy, these CLL patients therefore would be expected to be more sensitive to the treatment with mTOR inhibitors.

// Stephane Saint-Georges 1,2 , Maude Quettier 1,2 , Marouane Bouyaba 3 , Stephanie Le Coquil 1,2 , Vanessa Lauriente 1,2 , Lionel Guittat 1,2 , Vincent Levy 3 , Florence Ajchenbaum-Cymbalista 1,2,4 , Nadine Varin-Blank 1,2 , Christine Le Roy 1,2,* and Dominique Ledoux 1,2,* 1 INSERM U978, Bobigny, France 2 Universite Paris 13, Sorbonne Paris Cite, Labex “Inflamex”, Bobigny, France 3 Assistance Publique-Hopitaux de Paris, Hopital Avicenne, Unite de Recherche Clinique, Bobigny, France 4 Assistance Publique-Hopitaux de Paris, Hopital Avicenne, Service d’Hematologie Biologique, Bobigny, France * These authors are co-senior authors Correspondence to: Christine Le Roy, email: // Nadine Varin-Blank, email: // Keywords : CLL, lymphadenopathy, B-cell receptor, CXCR4/CXCR5, protein kinase D Received : December 23, 2015 Accepted : April 16, 2016 Published : April 26, 2016 Abstract In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser 324/325 . Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro . In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

// Burcu Yigit 1 , Peter J. Halibozek 1 , Shih-Shih Chen 2 , Michael S. O’Keeffe 1 , Jon Arnason 3 , David Avigan 3 , Valter Gattei 4 , Atul Bhan 5 , Osman Cen 6 , Richard Longnecker 6 , Nicholas Chiorazzi 2 , Ninghai Wang 1 , Pablo Engel 7 , Cox Terhorst 1 1 Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2 Karches Center for Chronic Lymphocytic Leukemia Research, The Feinstein Institute for Medical Research, Manhasset, NY, USA 3 Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 4 Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy 5 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 6 Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 7 Immunology Unit, Department of Cell Biology, Immunology and Neurosciences, Medical School, University of Barcelona, Barcelona, Spain Correspondence to: Burcu Yigit, email: byigit@bidmc.harvard.edu Cox Terhorst, email: cterhors@bidmc.harvard.edu Keywords: CLL, TCL1-192, SLAMF6, BCR, ibrutinib Received: January 29, 2016 Accepted: March 07, 2016 Published: March 25, 2016 ABSTRACT The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220 + CD5 + Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo , we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors.

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors. We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 – 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 – 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2′-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function. PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib.

CLL remains an incurable disease in spite of the many new compounds being studied. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the influence of stromal cells, key components of the CLL microenvironment, on the response of CLL cells to ATO. Bone marrow stromal cells induced CLL cell resistance to 2 μM ATO and led to activation of Lyn, ERK, PI3K and PKC, as well as NF-κB and STAT3. Mcl-1, Bcl-xL, and Bfl-1 were also upregulated after the co-culture. Inhibition experiments indicated that PI3K and PKC were involved in the resistance to ATO induced by stroma. Moreover, idelalisib and sotrastaurin, specific inhibitors for PI3Kδ and PKCβ, respectively, inhibited Akt phosphorylation, NF-κB/STAT3 activation and Mcl-1 upregulation, and rendered cells sensitive to ATO. Mcl-1 was central to the mechanism of resistance to ATO, since: 1) Mcl-1 levels correlated with the CLL cell response to ATO, and 2) blocking Mcl-1 expression or function with specific siRNAs or inhibitors overcame the protecting effect of stroma. We have therefore identified the mechanism involved in the CLL cell resistance to ATO induced by bone marrow stroma and show that idelalisib or sotrastaurin block this mechanism and restore sensibility to ATO. Combination of ATO with these inhibitors may thus constitute an efficient treatment for CLL.

CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2−/−IL2Rγc−/− mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL.

Most tumors circumvent telomere-length imposed replicative limits through expression of telomerase, the reverse transcriptase that maintains telomere length. Substantial evidence that AKT activity is required for telomerase activity exists, indicating that AKT inhibitors may also function as telomerase inhibitors. This possibility has not been investigated in a clinical context despite many clinical trials evaluating AKT inhibitors. We tested if Perifosine, an AKT inhibitor in clinical trials, inhibits telomerase activity and telomere maintenance in tissue culture and orthotopic xenograft models as well as in purified CLL samples from a phase II Perifosine clinical trial. We demonstrate that Perifosine inhibits telomerase activity and induces telomere shortening in a wide variety of cell lines in vitro, though there is substantial heterogeneity in long-term responses to Perifosine between cell lines. Perifosine did reduce primary breast cancer orthotopic xenograft tumor size, but did not impact metastatic burden in a statistically significant manner. However, Perifosine reduced telomerase activity in four of six CLL patients evaluated. Two of the patients were treated for four to six months and shortening of the shortest telomeres occurred in both patients' cells. These results indicate that it may be possible to repurpose Perifosine or other AKT pathway inhibitors as a novel approach to targeting telomerase.

Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E-/- mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors.

// Wojciech Wojtowicz 1 , Angelika Chachaj 2 , Andrzej Olczak 3 , Adam Ząbek 1 , Elzbieta Piątkowska 4 , Justyna Rybka 5 , Aleksandra Butrym 6, 7 , Monika Biedron 5 , Grzegorz Mazur 6 , Tomasz Wrobel 5 , Andrzej Szuba 2 and Piotr Mlynarz 1 1 Wroclaw University of Technology, Department of Bioorganic Chemistry, Wroclaw, Poland 2 Wroclaw Medical University, Department of Angiology, Wroclaw, Poland 3 Opole University of Technology, Faculty of Electrical Engineering, Automatic Control and Informatics, Opole, Poland 4 Wroclaw Research Centre EIT+, Wroclaw, Poland 5 Department of Haematology, Blood Neoplasms, and Bone Marrow Transplantation, Wroclaw Medical University, Wroclaw, Poland 6 Department of Internal Medicine, Wroclaw Medical University, Wroclaw, Poland 7 Department of Physiology, Wroclaw Medical University, Wroclaw, Poland Correspondence to: Piotr Mlynarz, email: piotr.mlynarz@pwr.edu.pl Keywords: metabolomics; haematological malignancies; nHL; AML; CLL Received: January 24, 2018 Accepted: April 07, 2018 Published: May 11, 2018 ABSTRACT Haematological malignancies are a frequently diagnosed group of neoplasms and a significant cause of cancer deaths. The successful treatment of these diseases relies on early and accurate detection. Specific small molecular compounds released by malignant cells and the simultaneous response by the organism towards the pathological state may serve as diagnostic/prognostic biomarkers or as a tool with relevance for cancer therapy management. To identify the most important metabolites required for differentiation, an 1 H NMR metabolomics approach was applied to selected haematological malignancies. This study utilized 116 methanol serum extract samples from AML (n= 38), nHL (n= 26), CLL (n= 21) and HC (n= 31). Multivariate and univariate data analyses were performed to identify the most abundant changes among the studied groups. Complex and detailed VIP-PLS-DA models were calculated to highlight possible changes in terms of biochemical pathways and discrimination ability. Chemometric model prediction properties were validated by receiver operating characteristic (ROC) curves and statistical analysis. Two sets of eight important metabolites in HC/AML/CLL/nHL comparisons and five in AML/CLL/nHL comparisons were selected to form complex models to represent the most significant changes that occurred.

Chemoimmunotherapy regimens have been the standard first-line therapy for patients with chronic lymphocytic leukemia (CLL). For young, fit patients the standard of care is combination of fludarabine, cyclophosphamide, and rituximab (FCR). Based on the preclinical work demonstrating that bendamustine combined with fludarabine resulted in increased DNA damage, we designed a phase I-II clinical trial with fludarabine, bendamustine, and rituximab (FBR) for patients with relapsed/refractory CLL. Treatment consisted of fludarabine 20 mg/m2 daily x 3 days and rituximab 375-500 mg/m2 x 1 day. Phase I included bendamustine at increasing doses of 20, 30, 40, or 50 mg/m2 daily x 3 days; phase II was with FR, and B at the selected dose. DNA damage response (H2AX phosphorylation) was evaluated in a subset of patients. Fifty-one patients were enrolled. The median age was 62 years; median number of prior therapies was 2; 40% had del(11q); and 41 patients had received prior FCR-based therapies. Hematologic toxicity was more common in ≥40 mg/m2 dose cohorts. Maximum tolerated dose (MTD) was not identified. Bendamustine-elicited H2AX phosphorylation was not dose-dependent, but markedly increased after fludarabine. We identified bendamustine 30 mg/m2 as the safe dose for phase II. The overall response rate (ORR) was 67% with 36% complete response (CR) / CR with incomplete count recovery (CRi). Younger patients (

Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells.

Aim of the study Flow cytometry has an important role in diagnosis and classification of B-cell lymphoproliferative disorders (BCLPDs). However, in distinguishing chronic lymphocytic leukemia (CLL) from small lymphocytic lymphoma (SLL) only clinical criteria are available so far. Aim of the study was to determine differences in the expression of common B cell markers (CD22, CD79b and CD20) on the malignant lymphocytes in the peripheral blood samples of CLL and SLL patients. Material and methods Peripheral blood samples of 56 CLL and 11 SLL patients were analyzed by 5-color flow cytometry on the CD45/CD19/CD5 gate for CD22, CD79b and CD20. Results In the samples collected from the CLL patients, CD22 expression was detected in only 20% of patients in the low pattern, while in SLL patients the expression was medium and present in 90.9% of patients (p < 0.0001). For CD79b expression, statistical significance is reached both in the expression pattern, which was low/medium for CLL and high for SLL, and expression level (p = 0.006). The expression of CD20 was counted as the CD20/CD19 ratio. The average ratio was 0.512 in the CLL patients vs. 0.931 in the SLL patients (p = 0.0001). Conclusions The pattern of expression and expression level of CD22, CD79b and CD20 in peripheral blood could be used for distinguishing SLL from CLL patients.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. Although promising new therapies for this incurable disease are being tested in clinical trials, the therapeutic relevance of metabolic rewiring in chronic lymphocytic leukemia (CLL) is poorly understood. The aim of this study was to identify targetable metabolic differences in primary CLL lymphocytes by the use of Dasatinib. Dasatinib is a multi-tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML) and is being tested in clinical trials for several cancers including CLL. This drug has been shown to be beneficial to CML patients suffering from diabetes by reducing their glucose plasma levels. In keeping with this previous observation, we report that Dasatinib induced glucose use while reducing lactate production, suggesting that this tyrosine kinase inhibitor decreases aerobic glycolysis and shifts glucose use in primary CLL lymphocytes. Our results suggest that primary CLL lymphocytes (independently of traditional prognostic factors) can be stratified in two subsets by their sensitivity to Dasatinib in vitro. Increased glucose use induced by Dasatinib or by inhibition of mitochondrial respiration was not sufficient to sustain survival and ATP levels in CLL samples sensitive to Dasatinib. The two subsets of primary CLL lymphocytes are characterized as well by a differential dependency on mitochondrial respiration and the use of anabolic or catabolic processes to cope with induced metabolic/energetic stress. Differential metabolic reprogramming between subsets is supported by the contrasting effect on the survival of Dasatinib treated CLL lymphocytes with pharmacological inhibition of two master metabolic regulators (mTorc1 and AMPK) as well as induced autophagy. Alternative metabolic organization between subsets is further supported by the differential basal expression (freshly purified lymphocytes) of active AMPK, regulators of glucose metabolism and modulators of AKT signaling. The contrasting metabolic features revealed by our strategy could be used to metabolically target CLL lymphocyte subsets creating new therapeutic windows for this disease for mTORC1 or AMPK inhibitors. Indeed, we report that Metformin, a drug used to treat diabetes was selectively cytotoxic to Dasatinib sensitive samples. Ultimately, we suggest that a similar strategy could be applied to other cancer types by using Dasatinib and/or relevant tyrosine kinase inhibitors.

Genomic complexity identified by chromosome banding analysis (CBA) predicts a worse clinical outcome in CLL patients treated either with standard or new treatments. Herein, we analyzed the clinical impact of complex karyotypes (CK) with or without high-risk FISH deletions (ATM and/or TP53, HR-FISH) in a cohort of 1045 untreated MBL/CLL patients. In all, 99/1045 (9.5%) patients displayed a CK. Despite ATM and TP53 deletions were more common in CK (25% vs 7%; P < 0.001; 40% vs 5%; P < 0.001, respectively), only 44% (40/90) patients with TP53 deletions showed a CK. CK group showed a significant higher two-year cumulative incidence of treatment (48% vs 20%; P < 0.001), as well as a shorter overall survival (OS) (79 mo vs not reached; P < 0.001). When patients were categorized regarding CK and HR-FISH, those with both characteristics showed the worst median OS (52 mo) being clearly distinct from those non-CK and non-HR-FISH (median not reached), but no significant differences were detected between cases with only CK or HR-FISH. Both CK and TP53 deletion remained statistically significant in the multivariate analysis for OS. In conclusion, CK group is globally associated with advanced disease and poor prognostic markers. Further investigation in larger cohorts with CK lacking HR-FISH is needed to elucidate which mechanisms underlie the poor outcome of this subgroup., This work has been supported by the following grants: PI11/01621, PI15/00437, RD12/0036/0044, RD12/0036/0069 and PT13/0010/0005 FEDER, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; 2014/SGR585 from Generalitat de Catalunya.

// Hilmar Quentmeier 1 , Claudia Pommerenke 1 , Ole Ammerpohl 2 , Robert Geffers 3 , Vivien Hauer 1 , Roderick AF MacLeod 1 , Stefan Nagel 1 , Julia Romani 1 , Emanuela Rosati 4 , Anders Rosen 5 , Cord C Uphoff 1 , Margarete Zaborski 1 , Hans G Drexler 1 1 Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 2 Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany 3 Genome Analytics Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany 4 Department of Experimental Medicine, Bioscience and Medical Embryology Section, University of Perugia, Perugia, Italy 5 Department of Clinical and Experimental Medicine, Division of Cell Biology, Linkoping University, Linkoping, Sweden Correspondence to: Hilmar Quentmeier, email: hqu@dsmz.de Keywords: CD5, cell lines, CLL, clonal evolution, subclones Received: June 07, 2016 Accepted: August 15, 2016 Published: August 23, 2016 ABSTRACT Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin ( IG ) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5 + , two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies.

Elisabeth Walsby 1 , Lawrence Pearce 1 , Alan K. Burnett 1 , Chris Fegan 1 , Chris Pepper 1 1 Department of Haematology, Institute of Cancer and Genetics, Cardiff University, School of Medicine, Cardiff, UK Received: May 2, 2012; Accepted: May 16, 2012; Published: May 19, 2012; Keywords: Hsp90, CLL, apoptosis, synergy, NF-κB Correspondence: Elisabeth Walsby, email: // // Abstract Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of multiple over-expressed signaling proteins that promote growth and survival in cancer cells. Chronic lymphocytic leukaemia (CLL) is characterized by increased expression of several Hsp90 client proteins making it a potentially susceptible to Hsp90 inhibition. In this study we showed that the novel Hsp90 inhibitor NVP-AUY922-AG was cytotoxic to primary CLL cells in vitro (LD50=0.18µM±0.20). Importantly, its toxicity was preserved under cytoprotective co-culture conditions that rendered fludarabine ineffective. At the molecular level, NVP-AUY922-AG depleted the expression of multiple Hsp90 client proteins including Akt and activators of NF-κB, IKKα and IKKβ. Consistent with this inhibition profile, NVP-AUY922-AG resulted in decreased transcription of the NF-B target genes MCL1, CFLAR, BIRC5. In contrast, fludarabine significantly induced the transcription of MCL1 and BIRC5. Given the anti-apoptotic nature of these genes and the role they play in fludarabine resistance, we considered that the combination of NVP-AUY922-AG with fludarabine might resensitize CLL cells to the effects of fludarabine. In keeping with this hypothesis, the combination of NVP-AUY922-AG and fludarabine was highly synergistic (mean CI=0.110.06) and this synergy was enhanced in co-culture (mean CI=0.06±0.08). Furthermore, the combination maintained the decrease in MCL1, CFLAR and BIRC5 transcription suggesting that the ability of NVP-AUY922-AG to modulate expression of these genes may contribute to the efficacy of this drug under cytoprotective co-culture conditions and for its remarkable synergy with fludarabine. Taken together these findings indicate that Hsp90 inhibition is an attractive therapeutic strategy in CLL.

// Stefania Fiorcari 1, * , Rossana Maffei 1, * , Valentina Audrito 2 , Silvia Martinelli 1 , Elisa ten Hacken 3 , Patrizia Zucchini 1 , Giulia Grisendi 1 , Leonardo Potenza 1 , Mario Luppi 1 , Jan A. Burger 3 , Silvia Deaglio 2 , Roberto Marasca 1 1 Hematology Unit, Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, Modena, Italy 2 Department of Medical Sciences, University of Turin and Human Genetics Foundation, Turin, Italy 3 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA * These authors have contributed equally to this work Correspondence to: Roberto Marasca, email: roberto.marasca@unimore.it Stefania Fiorcari, email: stefania.fiorcari@unimore.it Keywords: CLL, ibrutinib, microenvironment, nurse-like cells, immune modulation Received: June 08, 2016 Accepted: August 15, 2016 Published: September 01, 2016 ABSTRACT In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton’s tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

Patients with chronic lymphocytic leukemia (CLL) harboring TP53 aberrations (TP53abs; chromosome 17p deletion and/or TP53 mutation) exhibit an unfavorable clinical outcome. Chromosome 8 abnormalities, namely losses of 8p (8p-) and gains of 8q (8q+) have been suggested to aggravate the outcome of patients with TP53abs. However, the reported series were small, thus hindering definitive conclusions. To gain insight into this issue, we assessed a series of 101 CLL patients harboring TP53 disruption. The frequency of 8p- and 8q+ was 14.7% and 17.8% respectively. Both were associated with a significantly (P < 0.05) higher incidence of a complex karyotype (CK, ≥3 abnormalities) detected by chromosome banding analysis (CBA) compared to cases with normal 8p (N-8p) and 8q (N-8q), respectively. In univariate analysis for 10-year overall survival (OS), 8p- (P = 0.002), 8q+ (P = 0.012) and CK (P = 0.009) were associated with shorter OS. However, in multivariate analysis only CK (HR = 2.47, P = 0.027) maintained independent significance, being associated with a dismal outcome regardless of chromosome 8 abnormalities. In conclusion, our results highlight the association of chromosome 8 abnormalities with CK amongst CLL patients with TP53abs, while also revealing that CK can further aggravate the prognosis of this aggressive subgroup., This work has been supported by the following grants: PI11/01621, PI15/00437, RD09/0076/00036, RD12/0036/0044, RD12/0036/0069 and PT13/0010/0005 FEDER, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; 2014/SGR585 from Generalitat de Catalunya; “Xarxa de Bancs de tumors” sponsored by Pla Director d'Oncologia de Catalunya (XBTC) and Fundació La Caixa; the Swedish Cancer Society, the Swedish Research Council, the Lion's Cancer Research Foundation, and Selander's Foundation, Uppsala; H2020 “AEGLE, An analytics framework for integrated and personalized healthcare services in Europe” by the EU.

// Francesca Cattaneo 1 , Laura Patrussi 1 , Nagaja Capitani 1, 2 , Federica Frezzato 3 , Mario Milco D’Elios 2 , Livio Trentin 3 , Gianpietro Semenzato 3 , Cosima T. Baldari 1 1 Department of Life Sciences, University of Siena, Siena, Italy 2 Department of Experimental and Clinical Medicine, University of Florence, Firenze, Italy 3 Department of Medicine, University of Padua, Padova, Italy Correspondence to: Cosima T. Baldari, email: cosima.baldari@unisi.it Francesca Cattaneo, email: francesca.cattaneo@unisi.it Keywords: transcriptional regulation, p66Shc, STAT4, lisofylline, CLL Received: April 15, 2016 Accepted: July 19, 2016 Published: August 01, 2016 ABSTRACT p66Shc attenuates mitogenic, prosurvival and chemotactic signaling and promotes apoptosis in lymphocytes. Consistently, p66Shc deficiency contributes to the survival and trafficking abnormalities of chronic lymphocytic leukemia (CLL) B cells. The mechanism of p66shc silencing in CLL B cells is methylation-independent, at variance with other cancer cell types. Here we identify STAT4 as a novel transcriptional regulator of p66Shc in B cells. Chromatin immunoprecipitation and reporter gene assays showed that STAT4 binds to and activates the p66shc promoter. Silencing or overexpression of STAT4 resulted in a co-modulation of p66Shc. IL-12-dependent STAT4 activation caused a coordinate increase in STAT4 and p66Shc expression, which correlated with enhanced B cell apoptosis. Treatment with the STAT4 inhibitor lisofylline reverted partly this effect, suggesting that STAT4 phosphorylation is not essential for but enhances p66shc transcription. Additionally, we demonstrate that CLL B lymphocytes have a STAT4 expression defect which partly accounts for their p66Shc deficiency, as supported by reconstitution experiments. Finally, we show that p66Shc participates in a positive feedback loop to promote STAT4 expression. These results provide new insights into the mechanism of p66Shc expression in B cells and its defect in CLL, identifying the STAT4/IL-12 pathway as a potential therapeutic target in this neoplasia.

Dysfunctional apoptotic machinery is a hallmark feature of chronic lymphocytic leukemia (CLL). Accordingly, targeting apoptosis regulators has been proven a rational approach for CLL treatment. We show that CLL lymphocytes express high levels of XIAP, cIAP1, and cIAP2 compared to normal lymphocytes. Smac mimetic, Smac066, designed to bind to BIR3-domain of IAPs, induce apoptosis in primary CLL cells (n=71; p

B-cell chronic lymphocytic leukemia (CLL) is characterized by differential BCR\ud signaling and autoimmune complications. Complement modulates B-cell function\ud via C3d and CD21 cross-linked to the B-cell receptor (BCR). We hypothesized that\ud CD21 contributes to BCR signaling and participates in the autoimmunity associated\ud with CLL. We analyzed CD21 expression on 106 CLL patient samples and matched\ud serum from 50 patients for the presence of soluble CD21 and autoantibodies to CR2,\ud CR1, MCP and FH. CD21 expression on CLL B-cells was significantly lower than that\ud expressed on B-cells from age-matched controls (P < 0.0001) and was inversely\ud correlated with soluble CD21 (r2 = –0.41). We found no evidence of autoantibody to\ud any complement regulator. Low CD21 expression correlated to prognostic subsets\ud of CLL patients, i.e. cases with unmutated IGHV genes (P = 0.0006), high CD38\ud (P = 0.02) and high ZAP70 expression (P = 0.0017). Low CD21 expression was\ud inversely correlated to the levels of phosphotyrosine induced in CLL cells following\ud BCR ligation with αIgM (r2=–0.21). Importantly, lower CD21 expression was also\ud predictive for reduced overall survival (P = 0.005; HR = 2.7). In conclusion, we\ud showed that reduced expression of CD21 on CLL B-cells appears functionally relevant\ud and was associated with poor clinical outcomes.