Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer.

Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC50. ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However, a more comprehensive index composed of Mcl-1, Bcl-xL, Bim, Bak and Noxa protein or mRNA expression correlates well with Mcl-1 sensitivity in TNBC and can also predict Mcl-1 dependency in non-small cell lung cancer cell lines.

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16.

AIMS: To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species. METHODS AND RESULTS: Amycolatopsis genomic DNA was screened by PCR for the glycopeptide, Type-II (aromatic) polyketide and ansamycin biosynthetic gene clusters. Amycolatopsis sp. strain UM16, which exhibits weak antitubercular activity, was shown to have the glycopeptide oxyB gene and the Type-II (aromatic) polyketide-synthase KSalpha-KSbeta tandem gene pair, but not the AHBA synthase gene. The ristocetin (glycopeptide) producer, Amycolatopsis lurida NRRL 2430(T), was shown to have the oxyB gene and the Type-II polyketide-synthase KSalpha-KSbeta tandem gene pair. Amycolatopsis alba NRRL 18532(T) was shown to have the glycopeptide oxyB gene and the AHBA synthase gene. Phylogenetic analyses using Amycolatopsis oxyB and KSalpha-KSbeta gene sequences were conducted. CONCLUSIONS: Amycolatopsis sp. strain UM16 appears to have the biosynthetic potential to produce glycopeptide and Type-II polyketide antibiotics, but not ansamycins. The potential to synthesize aromatic polyketides may be more widely distributed in Amycolatopsis than is currently recognized. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening is a very useful tool for rapidly identifying the biosynthetic potential of an antibiotic-producing actinomycete isolate. Advanced knowledge of the type of antibiotic(s) produced will allow appropriate methods to be selected for antibiotic purification.