Targeting the Apoptotic Pathway by TW-37, a Novel Bcl-2 Family Small Molecule Inhibitor, In CLL Primary Samples

Impaired apoptosis is a hallmark of CLL cells, in association with overexpression of antiapoptotic Bcl-2 family members, including Bcl-2 and Mcl-1. Several compounds and antisense molecules that interfere with the Bcl-2 family have been proposed for the therapy of CLL, and some are already at clinical trials. These studies have shown that high levels of Mcl-1 may explain resistance to some of these compounds. Moreover, Mcl-1 has been related to BCR signaling and prolonged survival of CLL cells, while MCL-1 expression is an adverse prognosis marker. Previous gene expression studies from our laboratory have shown a heterogeneous expression of the different members of the Bcl-2 family, with subsets of cases showing increased expression. TW-37 is a novel Bcl-2 family small molecule inhibitor that derives from the natural compound gossypol and binds to the BH3-binding groove of Bcl-2, Bcl-X_L and interestingly also Mcl-1.Therefore, we aimed at studying the sensitivity of primary CLL samples to TW-37.

Forty-three peripheral blood samples were collected at diagnosis by the Tumor Bank unit at CNIO and processed in order to obtain either peripheral blood mononuclear cells (PBMCs) or purified B cells. Sensitivity to the compound was analyzed by EC50 calculations using the Cell Titer Glo® commercial kit from Promega. Mcl-1 antibody used was purchased from Santa Cruz (S-19). Expression profiling (Agilent microarray) were normalized and preprocessed using GEPAS utility available at http://gepas.bioinfo.cipf.es/. Apoptosis was measured by AnnexinV/ PI staining using flow cytometry.

The small molecule Bcl-2 inhibitor TW-37 was tested in a first series of PBMC CLL primary samples. EC50 values in the low nanomolar range (from 32.82 to 753.1 nM) were obtained. CLL cases with 17p loss showed a lower TW-37 sensitivity. When cases with 17p deletions were excluded from the study, there was a tendency of unmutated-CLL cases to be more sensitive to TW-37 (p= 0.07; n=23). TW-37 was tested on a second series of purified B cells and an inverse correlation between Mcl-1 protein levels and response to TW-37 (Pearson= −0.5; n=9) was observed. Consistently with what is known on the mechanism of action of the compound, TW-37 induced apoptosis in sensitive samples in a time and dose dependent manner. Using gene expression analysis, we identified a group of genes with higher expression in TW-37 resistant samples. This group included GADD45B, CXCL17, VAV2 and PKCQ (BCR signaling) and PIK3CB (p100β subunit of PI3K).

CLL samples with p53 pathway integrity were sensitive to Bcl-2-family inhibition using the TW-37 compound. This drug induced apoptosis in a time and dose dependent manner. Sensitivity was associated with naive IGHV genes, and higher levels of expression of Mcl-1, a potential biomarker of TW-37 sensitivity. Moreover, resistance to this compound seems to be related to differential expression of a gene signature that includes CXCL17, VAV2 and PKCQ; this signature might reflect the influence of the microenvironment and/or an exacerbated BCR activation.

Garcia-Marco: ROCHE: Consultancy, Honoraria, Research Funding.