Ataxia-telangiectasia mutated interacts with Parkin and induces mitophagy independent of kinase activity. Evidence from mantle cell lymphoma.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity,is frequently altered in human cancers including mantle cell lymphoma (MCL). Loss of ATM protein is linked to accumulation of nonfunctional mitochondria and defective mitophagy, in both murine thymocytes and in A-T cells. However, the mechanistic role of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that FCCP-induced mitophagy in MCL and other cancer cell lines is dependent on ATM but independent of its kinase function. While Granta-519 MCL cells possess single copy and kinase dead ATM and are resistant to FCCP-induced mitophagy, both Jeko-1 and Mino cells are ATM proficient and induce mitophagy. Stable knockdown of ATM in Jeko-1 and Mino cells conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mROS. ATM interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner. Knockdown of ATM in HeLa cells resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132 suggesting that ATM-Parkin interaction is important for Parkin stability. Neither loss of ATM kinase activity in primary B cell lymphomas nor inhibition of ATM kinase in MCL, A-T and HeLa cell lines mitigated FCCP or CCCP-induced mitophagy suggesting that ATM kinase activity is dispensable for mitophagy. Malignant B-cell lymphomas without detectable ATM, Parkin, Pink1, and Parkin-Ub ser65 phosphorylation were resistant to mitophagy, providing the first molecular evidence of ATM's role in mitophagy in MCL and other B-cell lymphomas.

CXCR5+CD8+ T cells are a distinct functional subset with an antitumor activity.

CXCR5 mediates homing of both B and follicular helper T (TFH) cells into follicles of secondary lymphoid organs. We found that CXCR5+CD8+ T cells are present in human tonsils and follicular lymphoma, inhibit TFH-mediated B cell differentiation, and exhibit strong cytotoxic activity. Consistent with these findings, adoptive transfer of CXCR5+CD8+ T cells into an animal model of lymphoma resulted in significantly greater antitumor activity than CXCR5-CD8+ T cells. Furthermore, RNA-Seq-based transcriptional profiling revealed 77 differentially expressed genes unique to CXCR5+CD8+ T cells. Among these, a signature comprised of 33 upregulated genes correlated with improved survival in follicular lymphoma patients. We also showed that CXCR5+CD8+ T cells could be induced and expanded ex vivo using IL-23 plus TGF-ß, suggesting a possible strategy to generate these cells for clinical application. In summary, our study identified CXCR5+CD8+ T cells as a distinct T cell subset with ability to suppress TFH-mediated B cell differentiation, exert strong antitumor activity, and confer favorable prognosis in follicular lymphoma patients.

Discovery and Optimization of Novel Hydrogen Peroxide Activated Aromatic Nitrogen Mustard Derivatives as Highly Potent Anticancer Agents.

We describe several new aromatic nitrogen mustards with various aromatic substituents and boronic esters that can be activated with H2O2 to efficiently cross-link DNA. In vitro studies demonstrated the anticancer potential of these compounds at lower concentrations than those of other clinically used chemotherapeutics, such as melphalan and chlorambucil. In particular, compound 10, bearing an amino acid ester chain, is selectively cytotoxic toward breast cancer and leukemia cells that have inherently high levels of reactive oxygen species. Importantly, 10 was 10-14-fold more efficacious than melphalan and chlorambucil for triple-negative breast-cancer (TNBC) cells. Similarly, 10 is more toxic toward primary chronic-lymphocytic-leukemia cells than either chlorambucil or the lead compound, 9. The introduction of an amino acid side chain improved the solubility and permeability of 10. Furthermore, 10 inhibited the growth of TNBC tumors in xenografted mice without obvious signs of general toxicity, making this compound an ideal drug candidate for clinical development.

The Stromal Microenvironment Modulates Mitochondrial Oxidative Phosphorylation in Chronic Lymphocytic Leukemia Cells.

Peripheral blood chronic lymphocytic leukemia (CLL) cells are replicationally quiescent mature B-cells. In short-term cultures, supporting stromal cells provide a survival advantage to CLL cells by inducing transcription and translation without promoting proliferation. We hypothesized that the stromal microenvironment augments malignant B cells' metabolism to enable the cells to cope with their energy demands for transcription and translation. We used extracellular flux analysis to assess the two major energy-generating pathways, mitochondrial oxidative phosphorylation (OxPhos) and glycolysis, in primary CLL cells in the presence of three different stromal cell lines. OxPhos, measured as the basal oxygen consumption rate (OCR) and maximum respiration capacity, was significantly higher in 28 patients' CLL cells cocultured with bone marrow-derived NK.Tert stromal cells than in CLL cells cultured alone (P = .004 and <.0001, respectively). Similar OCR induction was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis) were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Analysis of CLL cells' metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement of a prosurvival phenotype. The stroma did not impact the proliferation index (Ki-67 staining) of CLL cells. Collectively, these data suggest that short-term interaction (≤24 hours) with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells.

B-cell Receptor Signaling Regulates Metabolism in Chronic Lymphocytic Leukemia.

Peripheral blood chronic lymphocytic leukemia (CLL) cells are quiescent but have active transcription and translation processes, suggesting that these lymphocytes are metabolically active. Based on this premise, the metabolic phenotype of CLL lymphocytes was investigated by evaluating the two intracellular ATP-generating pathways. Metabolic flux was assessed by measuring glycolysis as extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation as oxygen consumption rate (OCR) and then correlated with prognostic factors. Further, the impact of B-cell receptor signaling (BCR) on metabolism was determined by genetic ablation and pharmacological inhibitors. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in CLL cells. ECAR was consistently low, but OCR varied considerably in human patient samples (n = 45). Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IGHV, high ß2M levels, and higher Rai stage. Consistent with the association of ZAP 70 and IGHV unmutated status with active BCR signaling, genetic ablation of BCR mitigated OCR in malignant B cells. Similarly, knocking out PI3Kδ, a critical component of the BCR pathway, decreased OCR and ECAR. In concert, PI3K pathway inhibitors dramatically reduced OCR and ECAR. In harmony with a decline in metabolic activity, the ribonucleotide pools in CLL cells were reduced with duvelisib treatment. Collectively, these data demonstrate that CLL metabolism, especially OCR, is linked to prognostic factors and is curbed by BCR and PI3K pathway inhibition.Implications: This study identifies a relationship between oxidative phosphorylation in CLL and prognostic factors providing a rationale to therapeutically target these processes. Mol Cancer Res; 15(12); 1692-703. ©2017 AACR.

Hydrogen peroxide activated quinone methide precursors with enhanced DNA cross-linking capability and cytotoxicity towards cancer cells.

Quinone methide (QM) formation induced by endogenously generated H2O2 is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of H2O2-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with H2O2 and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves H2O2-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development.

Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells.

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.

Comparison of Acalabrutinib, A Selective Bruton Tyrosine Kinase Inhibitor, with Ibrutinib in Chronic Lymphocytic Leukemia Cells.

Purpose: Ibrutinib inhibits Bruton tyrosine kinase (BTK) by irreversibly binding to the Cys-481 residue in the enzyme. However, ibrutinib also inhibits several other enzymes that contain cysteine residues homologous to Cys-481 in BTK. Patients with relapsed/refractory or previously untreated chronic lymphocytic leukemia (CLL) demonstrate a high overall response rate to ibrutinib with prolonged survival. Acalabrutinib, a selective BTK inhibitor developed to minimize off-target activity, has shown promising overall response rates in patients with relapsed/refractory CLL. A head-to-head comparison of ibrutinib and acalabrutinib in CLL cell cultures and healthy T cells is needed to understand preclinical biologic and molecular effects.Experimental Design: Using samples from patients with CLL, we compared the effects of both BTK inhibitors on biologic activity, chemokine production, cell migration, BTK phosphorylation, and downstream signaling in primary CLL lymphocytes and on normal T-cell signaling to determine the effects on other kinases.Results: Both BTK inhibitors induced modest cell death accompanied by cleavage of PARP and caspase-3. Production of CCL3 and CCL4 chemokines and pseudoemperipolesis were inhibited by both drugs to a similar degree. These drugs also showed similar inhibitory effects on the phosphorylation of BTK and downstream S6 and ERK kinases. In contrast, off-target effects on SRC-family kinases were more pronounced with ibrutinib than acalabrutinib in healthy T lymphocytes.Conclusions: Both BTK inhibitors show similar biological and molecular profile in primary CLL cells but appear different on their effect on normal T cells. Clin Cancer Res; 23(14); 3734-43. ©2016 AACR.

A phase I-II trial of fludarabine, bendamustine and rituximab (FBR) in previously treated patients with CLL.

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 (<65 years) had significantly higher ORR (81% vs. 50%; p=0.038). The median progression-free survival was 19 months, and the median overall survival was 52.5 months. FBR is an effective and tolerable CIT regimen for patients with relapsed CLL.

Reactivation of Smac-mediated apoptosis in chronic lymphocytic leukemia cells: mechanistic studies of Smac mimetic.

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<0.0001), irrespective of prognostic markers. Apoptosis was mediated by diminished levels of IAPs (XIAP-p=0.02; cIAP-p<0.0001) and increased activation of caspases-8,-9,-3. The caspase-cleavage was in direct association with the levels of apoptosis (r2=0.8 for caspases-8,-9,-3). Correlative analysis revealed a direct relationship between reduction in IAPs and degree of apoptosis (r2=0.6 (XIAP); 0.5 (cIAP2)). There was a strong association between apoptosis, IAP-degradation, and concurrent caspase-activation. Pan-caspase inhibitor Z-Vad-fmk reversed the degradation of Mcl-1, but not IAPs suggesting that smac066 is selective to IAPs, however, Mcl-1 degradation is through caspase-mediated cleavage. Immunoprecipitation experiments revealed physical interaction between caspase-3 and XIAP that was disrupted by smac066. Importantly, XIAP and cIAP2 were markedly induced in bone-marrow and lymph-node microenvironments, providing a basis for IAP antagonists as anti-tumor agents in CLL. Smac066 synergized with ABT-737, revealing a mechanistic rationale to jointly target BH3 and BIR3 domains.

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia.

PURPOSE: Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. EXPERIMENTAL DESIGN: Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. RESULTS: The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. CONCLUSIONS: Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL.

Expression of executioner procaspases and their activation by a procaspase-activating compound in chronic lymphocytic leukemia cells.

Intrinsic and extrinsic apoptotic pathways converge to activate common downstream executioner caspases (caspase-3, -6, and -7), resulting in cell death. In chronic lymphocytic leukemia (CLL), neoplastic B cells evade apoptosis owing to the overexpression of survival proteins. We hypothesized that direct activation of procaspases could bypass the apoptosis resistance induced by the upstream prosurvival proteins. The procaspase-activating compounds (PAC-1), including B-PAC-1 (L14R8), convert inactive executioner procaspases to their active cleaved forms by chelation of labile zinc ions. Both at transcript and protein levels, primary CLL cells express high levels of latent procaspases (3, -7, and -9). B-PAC-1 treatment induced CLL lymphocyte death which was higher than that in normal peripheral blood mononuclear cells or B cells, and was independent of prognostic markers and microenvironmental factors. Mechanistically, B-PAC-1 treatment activated executioner procaspases and not other Zn-dependent enzymes. Exogenous zinc completely, and pancaspase inhibitors partially, reversed B-PAC-1-induced apoptosis, elucidating the zinc-mediated mechanism of action. The cell demise relied on the presence of caspase-3/7 but not caspase-8 or Bax/Bak proteins. B-PAC-1 in combination with an inhibitor of apoptosis protein antagonist (Smac066) synergistically induced apoptosis in CLL samples. Our investigations demonstrated that direct activation of executioner procaspases via B-PAC-1 treatment bypasses apoptosis resistance and is a novel approach for CLL therapeutics.

Regulation of Mcl-1 expression in context to bone marrow stromal microenvironment in chronic lymphocytic leukemia.

A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3ß at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment.

Reactive oxygen species (ROS) inducible DNA cross-linking agents and their effect on cancer cells and normal lymphocytes.

Reducing host toxicity is one of the main challenges of cancer chemotherapy. Many tumor cells contain high levels of ROS that make them distinctively different from normal cells. We report a series of ROS-activated aromatic nitrogen mustards that selectively kill chronic lymphocytic leukemia (CLL) over normal lymphocytes. These agents showed powerful DNA cross-linking abilities when coupled with H2O2, one of the most common ROS in cancer cells, whereas little DNA cross-linking was detected without H2O2. Consistent with chemistry observation, in vitro cytotoxicity assay demonstrated that these agents induced 40-80% apoptosis in primary leukemic lymphocytes isolated from CLL patients but less than 25% cell death to normal lymphocytes from healthy donors. The IC50 for the most potent compound (2) was ~5 µM in CLL cells, while the IC50 was not achieved in normal lymphocytes. Collectively, these data provide utility and selectivity of these agents that will inspire further and effective applications.

Phase I and extension study of clofarabine plus cyclophosphamide in patients with relapsed/refractory acute lymphoblastic leukemia.

BACKGROUND: Clofarabine is a nucleoside analogue with activity in children with acute lymphoblastic leukemia (ALL). Based on the hypothesis that clofarabine inhibits DNA repair after exposure to DNA-damaging agents, we designed a phase I and extension study to evaluate the combination of clofarabine and cyclophosphamide in adult patients with relapsed/refractory ALL. METHODS: The continual reassessment method (CRM) was used to define the maximum tolerated dose (MTD). RESULTS: Fifty patients with a median age of 30 years (range, 21-72 years) were enrolled, 30 of whom were part of the phase I group. Clofarabine 40 mg/m(2) intravenously daily × 3 days and cyclophosphamide 200 mg/m(2) intravenously every 12 hours × 3 days were established as the MTDs. Dose limiting toxicity (DLT) included diarrhea, transaminase elevations, and skin rashes. The response rate of the whole study group was 14%, including 10% of patients who achieved complete remission (CR) or CR without platelet recovery (CRp). Three responses occurred in patients with primary refractory disease. Early mortality (< 30 days) was 6%. The median duration of response was 69 days (range, 5-315 days). Median overall survival was about 3 months. Compared with day 1 (cyclophosphamide alone), H2AX phosphorylation was increased on day 2 when clofarabine and cyclophosphamide were administered as a couplet (n = 8). CONCLUSION: The combination of clofarabine plus cyclophosphamide at the doses used in this study in a group of heavily pretreated patients with ALL is only moderately effective. Other doses, alternative schedules, or a more favorable patient population may achieve better results.

Impact of bone marrow stromal cells on Bcl-2 family members in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world. High levels of Bcl-2 family anti-apoptotic proteins are responsible for apoptosis resistance. Besides anti-apoptotic proteins, the microenvironment provides substantial survival signals to CLL leukemic cells. However, in-depth knowledge on the role of individual Bcl-2 family members in the context of the microenvironment is still limited. We performed a comprehensive analysis of transcripts and proteins of 18 Bcl-2 family members using an "apoptosis array microfluidic card" in primary cells before and after stromal co-cultures. Our data showed that five of six anti-apoptotic members (excluding Bcl-b), two of three pro-apoptotic members (excluding Bok) and six of nine BH3-only members were present at detectable mRNA levels in CLL cells. Importantly, stromal-mediated extended survival of CLL cells was strongly associated with elevated global transcription. Upon co-culturing with stromal cells, there was an early response of an increase in anti- (2/5) and pro-apoptotic protein (3/8) transcripts on day 1, while an increase in anti-apoptotic proteins was observed on day 3, with no significant change in pro-apoptotic proteins. Our study revealed a differential pattern of expression of both transcripts and proteins following stromal co-cultures, proposing a significance of Bcl-2 family members in the stromal microenvironment.

DNA repair initiation induces expression of ribonucleotide reductase in human chronic lymphocytic leukemia cells.

Mammalian ribonucleotide reductase (RR) is a heterodimer enzyme responsible for maintaining levels of deoxynucleotides needed for DNA replication. The M2 subunit of RR is known to increase in tandem with progression of cells into S phase, whereas the M1 subunit is expressed at steady-state. Since the expression level of the M2 subunit increases because the cells need deoxyribonucleoside triphosphates (dNTPs) for replication, it is logical to hypothesize that the same increase will be seen during DNA repair. To test this, we used chronic lymphocytic leukemia (CLL) cells, which are replicationally quiescent and have low endogenous levels of RR and dNTPs. Cyclophosphamide was selected as the DNA damaging agent because of its clinical use in the treatment of CLL. DNA repair, measured by [(3)H]thymidine incorporation after 4 h treatment with 4-hydroperoxycyclophosphamide, increased in a dose-dependent manner at 3, 10 and 50 µM. The induction of DNA repair concomitantly increased the mRNA and protein levels of M2 subunit (median 1.6-fold; range 0.9-5.3). Maximum induction occurred at 10 µM after 4 h and correlated with [(3)H]thymidine incorporation (p = 0.02). In contrast, no change was observed in mRNA or protein levels of M1 subunit. We conclude that RR is regulated not only during replication but also during DNA repair, and in both cases M2 subunit expression is increased.

Targeting MET kinase with the small-molecule inhibitor amuvatinib induces cytotoxicity in primary myeloma cells and cell lines.

BACKGROUND: MET is a receptor tyrosine kinase that is activated by the ligand HGF and this pathway promotes cell survival, migration, and motility. In accordance with its oncogenic role, MET is constitutively active, mutated, or over-expressed in many cancers. Corollary to its impact, inhibition of MET kinase activity causes reduction of the downstream signaling and demise of cells. In myeloma, a B-cell plasma malignancy, MET is neither mutated nor over-expressed, however, HGF is increased in plasma or serum obtained from myeloma patients and this was associated with poor prognosis. The small-molecule, amuvatinib, inhibits MET receptor tyrosine kinase. Based on this background, we hypothesized that targeting the HGF/MET signaling pathway is a rational approach to myeloma therapy and that myeloma cells would be sensitive to amuvatinib. METHODS: Expression of MET and HGF mRNAs in normal versus malignant plasma cells was compared during disease progression. Cell death and growth as well as MET signaling pathway were assessed in amuvatinib treated primary myeloma cells and cell lines. RESULTS: There was a progressive increase in the transcript levels of HGF (but not MET) from normal plasma cells to refractory malignant plasma cells. Amuvatinib readily inhibited MET phosphorylation in primary CD138+ cells from myeloma patients and in concordance, increased cell death. A 48-hr amuvatinib treatment in high HGF-expressing myeloma cell line, U266, resulted in growth inhibition. Levels of cytotoxicity were time-dependent; at 24, 48, and 72 h, amuvatinib (25 µM) resulted in 28%, 40%, and 55% cell death. Consistent with these data, there was an amuvatinib-mediated decrease in MET phosphorylation in the cell line. Amuvatinib at concentrations of 5, 10, or 25 µM readily inhibited HGF-dependent MET, AKT, ERK and GSK-3-beta phosphorylation. MET-mediated effects were not observed in myeloma cell line that has low MET and/or HGF expression. CONCLUSIONS: These data suggest that at the cellular level MET/HGF pathway inclines with myeloma disease progression. Amuvatinib, a small molecule MET kinase inhibitor, is effective in inducing growth inhibition and cell death in myeloma cell lines as well as primary malignant plasma cells. These cytostatic and cytotoxic effects were associated with an impact on MET/HGF pathway.