Mitochondrial Respiration Correlates with Prognostic Markers in Chronic Lymphocytic Leukemia and Is Normalized by Ibrutinib Treatment.

Mitochondrial bioenergetics profiling, a measure of oxygen consumption rates, correlates with prognostic markers and can be used to assess response to therapy in chronic lymphocytic leukemia (CLL) cells. In this study, we measured mitochondrial respiration rates in primary CLL cells using respirometry to evaluate mitochondrial function. We found significant increases in mitochondrial respiration rates in CLL versus control B lymphocytes. We also observed amongst CLL patients that advanced age, female sex, zeta-chain-associated protein of 70 kD (ZAP-70+), cluster of differentiation 38 (CD38+), and elevated ß2-microglobulin (ß2-M) predicted increased maximal respiration rates. ZAP-70+ CLL cells exhibited significantly higher bioenergetics than B lymphocytes or ZAP-70- CLL cells and were more sensitive to the uncoupler, carbonyl cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP). Univariable and multivariable linear regression analysis demonstrated that ZAP-70+ predicted increased maximal respiration. ZAP-70+ is a surrogate for B cell receptor (BCR) activation and can be targeted by ibrutinib, which is a clinically approved Bruton's tyrosine kinase (BTK) inhibitor. Therefore, we evaluated the oxygen consumption rates (OCR) of CLL cells and plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4) levels from ibrutinib-treated patients and demonstrated decreased OCR similar to control B lymphocytes, suggesting that ibrutinib treatment resets the mitochondrial bioenergetics, while diminished CCL3/CCL4 levels indicate the down regulation of the BCR signaling pathway in CLL. Our data support evaluation of mitochondrial respiration as a preclinical tool for the response assessment of CLL cells.

Transcriptional Modulation by Idelalisib Synergizes with Bendamustine in Chronic Lymphocytic Leukemia.

: The phosphatidyl-inositol 3 kinase (PI3K) δ inhibitor, idelalisib (IDE), is a potent inhibitor of the B-cell receptor pathway and a novel and highly effective agent for the treatment of chronic lymphocytic leukemia (CLL). We evaluated the activities of IDE in comparison to bendamusine (BEN), a commonly used alkylating agent, in primary CLL cells ex vivo. In contrast to BEN, IDE was cytotoxic to cells from extensively-treated patients, including those with a deletion (del)17p. Cross-resistance was not observed between BEN and IDE, confirming their different modes of cytotoxicity. Marked synergy was seen between BEN and IDE, even in cases that were resistant to BEN or IDE individually, and those with deletion (del) 17p. CD40L/interleukin 4 (IL4) co-treatment mimicking the CLL microenvironment increased resistance to IDE, but synergy was retained. PI3Kδ-deficient murine splenic B cells were more resistant to IDE and showed reduced synergy with BEN, thus confirming the importance of functional PI3Kδ protein. Although IDE was observed to induce γH2AX, IDE did not enhance activation of the DNA damage response nor DNA repair activity. Interestingly, IDE decreased global RNA synthesis and was antagonistic with 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB), an inhibitor of transcription. These findings add to the increasingly complex cellular effects of IDE, and B cell receptor (BCR) inhibitors in general, in CLL.

EWS-FLI1 confers exquisite sensitivity to NAMPT inhibition in Ewing sarcoma cells.

Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.

Cross-resistance and synergy with bendamustine in chronic lymphocytic leukemia.

Bendamustine (BEN) has structural similarities to an alkylating agent and a nucleoside analog, and effective against tumor cells that are resistant to standard therapy. In this study we compared the activities of BEN against that of the alkylating agent, chlorambucil (CLB), and the nucleoside analogs, fludarabine (FLU) and deoxyadenosine/pentostatin (dADO/PEN), in primary chronic lymphocytic leukemia (CLL) cells in vitro. Cross-resistance was observed between BEN, CLB and FLU, with previously treated patients or those with a deletion 17p being most resistant. In contrast, some resistant CLL cells retained moderate sensitivity to dADO/PEN. Like FLU and CLB, BEN induced apoptosis through both the mitochondrial and death receptor pathways. There was a greater increase in DNA double-strand breaks (DSB) following FLU, as compared to BEN and CLB. Synergistic cytotoxicity was seen on combining BEN or CLB with FLU or dADO/PEN, but not when combining BEN with CLB. These results demonstrate that BEN acts as an alkylating agent, demonstrates cross-resistance to CLB and FLU and resistance to cells with a del 17p. Synergistic cytotoxic activity was seen between BEN and dADO/PEN suggesting that the combination of BEN and PEN should be evaluated in the clinic.

On-target effect of FK866, a nicotinamide phosphoribosyl transferase inhibitor, by apoptosis-mediated death in chronic lymphocytic leukemia cells.

PURPOSE: Chronic lymphocytic leukemia (CLL) remains incurable despite advances in therapy. In this study, we characterize the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibition by FK866 in primary CLL cells from patients with various clinical prognostic markers. EXPERIMENTAL DESIGN: CLL cells were treated with FK866 to assess viability by Annexin V/PI staining. Functional analysis of FK866 included time- and concentration-dependent evaluation of cellular NAD, ATP, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and apoptotic signaling. Chemosensitization potential by FK866 to fludarabine was also assessed. Prognostic markers were correlated with drug response. RESULTS: FK866 induced CLL cell death by depleting cellular NAD content by day 1, followed by a drop in ATP on day 2. We observed loss of MMP, ROS increase, and induction of apoptotic signaling at day 3. On-target activity of FK866 was confirmed by NAD-mediated rescue of NAD and ATP loss, apoptotic signaling, and viability. The response to FK866 was independent of most prognostic markers. Higher doses were required with short lymphocyte doubling time and positive CD38 status, whereas CLL cells resistant to fludarabine in vitro and from patients with del17p13.1 were equally sensitive to FK866. FK866 did not upregulate the p53-target p21, nor did the p53 activator Nutlin improve FK866-mediated cell death. Furthermore, fludarabine and FK866 were synergistic at clinically relevant concentrations. CONCLUSIONS: NAMPT inhibition by FK866 may be a potential treatment for CLL, including patients with del17p13.1 or other high-risk features. FK866 may complement standard agents to enhance their efficacy and/or allow dose reduction for improved tolerability.