Clinical responses to bendamustine in chronic lymphocytic leukemia (CLL) are highly heterogeneous and no specific markers to predict sensitivity to this drug have been reported. In order to identify biomarkers of response, we analyzed the in vitro activity of bendamustine and the gene expression profile in primary CLL cells. We observed that mRNA expression of CD69 (CD69) and ITGAM (CD11b) constitute the most powerful predictor of response to bendamustine. When we interrogated the predictive value of the corresponding cell surface proteins, the expression of the activation marker CD69 was the most reliable predictor of sensitivity to bendamustine. Importantly, a multivariate analysis revealed that the predictive value of CD69 expression was independent from other clinico-biological CLL features. We also showed that when CLL cells were co-cultured with distinct subtypes of stromal cells, an upregulation of CD69 was accompanied by a reduced sensitivity to bendamustine. In agreement with this, tumor cells derived from lymphoid tumor niches harbored higher CD69 expression and were less sensitive to bendamustine than their peripheral blood counterparts. Furthermore, pretreatment of CD69 high CLL cases with the B-cell receptor (BCR) pathway inhibitors ibrutinib and idelalisib decreased CD69 levels and enhanced bendamustine cytotoxic effect. Collectively, our findings indicate that CD69 could be a predictor of bendamustine response in CLL patients and the combination of clinically-tested BCR signaling inhibitors with bendamustine may represent a promising strategy for bendamustine low responsive CLL cases.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and-unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

Bendamustine is used in the treatment of chronic lymphocytic leukemia (CLL). Routes for bendamustine entry into target cells are unknown. This study aimed at identifying transporter proteins implicated in bendamustine uptake. Our results showed that hOCT1 is a bendamustine transporter, as bendamustine could cis-inhibit the uptake of a canonical hOCT1 substrate, with a Ki in the micromolar range, consistent with the EC50 values of the cytotoxicity triggered by this drug in HEK293 cells expressing hOCT1. hOCT1 polymorphic variants determining impaired bendamustine-transporter interaction, consistently reduced bendamustine cytotoxicity in HEK293 cells stably expressing them. Exome genotyping of the SLC22A1 gene, encoding hOCT1, was undertaken in a cohort of 241 CLL patients. Ex vivo cytotoxicity to bendamustine was measured in a subset of cases and shown to correlate with SLC22A1 polymorphic variants. In conclusion, hOCT1 is a suitable bendamustine transporter, thereby contributing to its cytotoxic effect depending upon the hOCT1 genetic variants expressed.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to be highly effective in patients with chronic lymphocytic leukemia (CLL) and is approved for CLL treatment. Unfortunately, resistance and intolerance to ibrutinib has been observed in several studies, opening the door for more specific BTK inhibitors. CC‐292 (spebrutinib) is a BTK inhibitor with increased specificity for BTK and less inhibition of other kinases. Our in vitro studies showed that CC‐292 potently inhibited B‐cell receptor signaling, activation, proliferation and chemotaxis of CLL cells. In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC‐292 reduced tumor load and normalized tumor‐associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC‐292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC‐292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.

Acadesine is a nucleoside analogue with known activity against B-cell malignancies. Herein, we showed that in mantle cell lymphoma (MCL) cells acadesine induced caspase-dependent apoptosis through turning on the mitochondrial apoptotic machinery. At the molecular level, the compound triggered the activation of the AMPK pathway, consequently modulating known downstream targets, such as mTOR and the cell motility-related vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation by acadesine was concomitant with a blockade of CXCL12-induced migration. The inhibition of the mTOR cascade by acadesine, committed MCL cells to enter in apoptosis by a translational downregulation of the antiapoptotic Mcl-1 protein. In contrast, Bcl-2 protein levels were unaffected by acadesine and MCL samples expressing high levels of Bcl-2 tended to have a reduced response to the drug. Targeting Bcl-2 with the selective BH3-mimetic agent ABT-199 sensitized Bcl-2 high MCL cells to acadesine. This effect was validated in vivo, where the combination of both agents displayed a more marked inhibition of tumor outgrowth than each drug alone. These findings support the notions that antiapoptotic proteins of the Bcl-2 family regulate MCL cell sensitivity to acadesine and that the combination of this agent with Bcl-2 inhibitors might be an interesting therapeutic option to treat MCL patients.

Next generation sequencing has provided a comprehensive understanding of the mutational landscape in chronic lymphocytic leukemia (CLL), and new drivers have been identified. Some of these drivers could be pharmacologically targeted to choose the most effective personalized therapy in each CLL patient. Areas covered: In this article, the authors uncover the potential role of new targeted therapies against the most recurrent mutations in CLL as well as the recently approved therapies. The authors also provide their expert opinion and give their perspectives for the future. Expert opinion: The development of more personalized therapies is of interest to clinicians as a system to enhance the duration of treatment response and to extend the survival and quality of life of CLL patients. The main challenge, however, will be to translate the preclinical results into the clinics. Therefore, the designing and execution of clinical trials focused on molecular drivers are the need of the hour.

Chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is characterized by the accumulation of monoclonal CD5+ B cells in the blood, bone marrow, lymph nodes, spleen and other lymphoid organs. Bendamustine hydrochloride, a cytotoxic agent comprising structural features of both an alkylating drug and a purine nucleoside analog, was recently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of CLL and indolent B cell non-Hodgkin's lymphoma (NHL). Although several recent clinical trials have documented the activity of bendamustine in CLL, the mechanisms of bendamustine resistance in CLL cells have not been yet elucidated. We analyzed the ex vivo cytotoxicity of bendamustine in cells from 48 CLL cases by incubating them with bendamustine 25 μM for 24 hours. Cell death was assessed by annexinV-PI labeling and cases were classified as bendamustine-resistant (cytotoxicity below 30%) or bendamustine-sensitive (cytotoxicity over 30%) cases. We found that sensitivity of CLL primary cells correlated with the mutational status of IGHV gene, being the IGHV-unmutated cases significantly more resistant to bendamustine (p<0.015) than those cases with mutated IGHV. Although the number of cases analyzed was low, no correlation with the mutational status of NOTCH1 or SF3B1 was observed. We compared the gene expression profiling of bendamustine-resistant and bendamustine-sensitive groups by performing a high-throughput analysis of basal gene expression in CLL cells with the U219 Affymetrix array. After a supervised analysis using the Rank Products method and a FDR below 0.001, we observed 393 up-regulated genes and 415 down-regulated genes in the bendamustine-resistant group. Annotation of the differentially expressed genes identified some well-known markers of prognosis in CLL, together with genes related to apoptosis regulation and B cell activation. One of the genes with a highest positive fold-change and most relevant according to its role in cell cycle progression, apoptosis and transformation was C-MYC. These results suggest that overexpression of genes related to proliferation and B-cell activation could be involved in CLL resistance to bendamustine therapy, which is known to be clinically more effective in indolent than in aggressive lymphomas. Further studies will assess whether this set of overexpressed genes might be useful as biomarkers to predict the sensitivity to bendamustine and open the door to new combinatorial strategies that might overcome resistance to bendamustine in CLL patients within the context of tailored medicine Citation Format: Eriong Lee-Verges, Arnau Montraveta, Magda Pinyol, Pedro Jares, Cristina Arimany-Nardi, Marta Aymerich, Neus Villamor, Marçal Pastor-Anglada, Armando Lopez-Guillermo, Patricia Perez-Galan, Gael Roue, Elias Campo, Dolors Colomer. Upregulation of B-cell activation genes and negative regulators of apoptosis determines resistance to bendamustine in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2013-993

Bendamustine hydrochloride is a cytotoxic agent that encompasses structural features of alkylating and nucleoside-derived drugs. This drug is currently used in the treatment of several haemotological malignancies; among them Chronic Lymphocytic Leukemia (CLL). CLL is the most common leukemia in Western countries and is characterized by the accumulation of monoclonal CD5+ B cells in the blood, bone marrow, lymph nodes, spleen and other organs. The pathways facilitating the uptake of bendamustine hydrochloride into target cells still remain poorly understood. Here we have addressed this issue by monitoring the interaction of the drug with selected plasma membrane transporter candidates, which might be relevant to bendamustine pharmacokinetics and pharmacodynamics. We have used a panel of HEK293 cells stably expressing selected drug transporter proteins to prove that bendamustine is able to interact with human Organic Cation Transporter 1 (hOCT1) with a Ki of 140+/-10 μM, as deduced from cis-inhibition studies using tritiated 1-methyl-4-phenylpyridinium (MPP+) as hOCT model substrate. Genetic variants known to modify substrate/transporter interactions, some of them with high allelic frequency in humans, were also tested and, in particular, hOCT1M420Del showed a significant decrease in its affinity for the drug (Ki value 234+/-24 μM, p<0.05 vs the wild type transporter). The whole panel of hOCT1 and hOCT1 allelic variants were also tested for bendamustine-induced cytotoxicity and data consistent with bendamustine being a hOCT1 substrate were obtained. This was based upon the finding that hOCT1 expression triggered increased sensitivity to the drug and variants known to decrease drug-transporter affinity interactions were also shown to induce resistance. hOCT1 is expressed in CLL cells and genotyping of a cohort of 125 CLL patients is now allowing the analysis of the relationships between particular genetic variants and ex vivo bendamustine-induced cytotoxicity, thus opening the possibility that hOCT1 genotyping might be a useful tool in optimizing bendamustine-based therapies. Citation Format: Cristina Arimany-Nardi, Arnau Montraveta, Eriong Lee-Vergés, Hermann Koepsell, Dolors Colomer, Marçal Pastor-Anglada. Human organic cation transporter 1 (hOCT1) as a mediator of bendamustine cytotoxic action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 885. doi:10.1158/1538-7445.AM2013-885