Christin Tse

Overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) is commonly associated with tumor maintenance, progression, and chemoresistance. We previously reported the discovery of ABT-737, a potent, small-molecule Bcl-2 family protein inhibitor. A major limitation of ABT-737 is that it is not orally bioavailable, which would limit chronic single agent therapy and flexibility to dose in combination regimens. Here we report the biological properties of ABT-263, a potent, orally bioavailable Bad-like BH3 mimetic (K(i)'s of <1 nmol/L for Bcl-2, Bcl-xL, and Bcl-w). The oral bioavailability of ABT-263 in preclinical animal models is 20% to 50%, depending on formulation. ABT-263 disrupts Bcl-2/Bcl-xL interactions with pro-death proteins (e.g., Bim), leading to the initiation of apoptosis within 2 hours posttreatment. In human tumor cells, ABT-263 induces Bax translocation, cytochrome c release, and subsequent apoptosis. Oral administration of ABT-263 alone induces complete tumor regressions in xenograft models of small-cell lung cancer and acute lymphoblastic leukemia. In xenograft models of aggressive B-cell lymphoma and multiple myeloma where ABT-263 exhibits modest or no single agent activity, it significantly enhances the efficacy of clinically relevant therapeutic regimens. These data provide the rationale for clinical trials evaluating ABT-263 in small-cell lung cancer and B-cell malignancies. The oral efficacy of ABT-263 should provide dosing flexibility to maximize clinical utility both as a single agent and in combination regimens.

We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg2+ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl2-containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg2+-dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays.

The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.