Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.     

Site-specific modification of single cysteine Pax3 mutants reveals reciprocal regulation of DNA binding activity of the paired and homeo domain

The mechanism by which the paired domain (PD) and the homeo domain (HD) act together in the intact Pax3 protein to recognize DNA is unclear and was studied in a Pax3 mutant (Pax3-CL) devoid of cysteines. Pax3-CL binds to PD (P6CON-P3OPT sites) and HD (P2, P1/2 sites) DNA site sequences with near wild-type activity but, contrary to Pax3, in a N-ethyl maleimide (NEM) insensitive fashion. The Pax3-CL backbone was used for cysteine scanning mutagenesis and for site-specific NEM modification. Five single cysteine replacements were independently introduced in the PD, while eight were inserted in the HD. NEM sensitivity of PD and HD DNA binding was investigated in DNA-binding competent mutants. In the PD mutant C82, NEM abrogated DNA binding by the PD but also abolished DNA binding by the Cys-less HD. Likewise, in the HD mutant V263C, NEM modification abrogated DNA binding not only by the HD, but also by the Cys-less PD. The transfer of NEM sensitivity to the PD seen in V263C was specific and not due to simple loss of HD DNA binding since alkylation of adjacent V265C and S268C, although impairing HD DNA binding did not affect PD DNA binding. Thus, the PD and HD do not function as independent DNA binding modules in Pax3 but seem functionally interdependent.(1)     

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-X_L is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-X_L inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-X_L. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-X_L for their survival and indicate Bcl-X_L inhibition as a potential therapeutic avenue in NSCLC.Lung cancer is the leading cause of cancer-related death in men and is expected to become the main cause of cancer death for women in the near future.^{1, 2} There is increasing evidence that cancer stem cells (CSCs) have a key role in drug resistance, tumor progression and metastasis in multiple tumor types, including lung cancer.³ Lung cancer stem cells (LCSCs) have been previously identified through different criteria including surface expression of CD133, c-kit or through functional properties such as selective drug survival, elevated aldehyde dehydrogenase (ALDH) activity, increased glycolysis and glycine/serine metabolism or low concentrations of reactive oxygen species and ATP.^{4, 5, 6, 7, 8, 9} Importantly, when inoculated into immunocompromised mice, LCSCs give rise to xenografts that histologically reproduce the tumor of origin, thus representing an improved model for in vivo testing of new targeted therapies.¹⁰ Several tumors express elevated levels of anti-apoptotic Bcl-2 family proteins such as Bcl-2, Bcl-X_L and Mcl-1, which affect the apoptotic threshold of neoplastic cells contributing to chemotherapy resistance.¹¹ Inhibition of anti-apoptotic Bcl-2 family members has been for long time regarded as a promising strategy to induce cancer cell death through approaches of increasing specificity. BH3 mimetics such as ABT-737, the related orally available ABT-263 (navitoclax) and the recently developed Bcl-2-selective inhibitor ABT-199 have been shown to exert an antitumor effect in preclinical and clinical settings either as single agents or in combination with conventional or targeted drugs.¹² Recently, a new role for Bcl-2 has emerged in acute myeloid leukemia (AML), where quiescent stem cells characterized by low levels of reactive oxygen species were found to overexpress Bcl-2 and rely on this factor for survival.¹³ Similarly, in chronic myeloid leukemia (CML), quiescent therapy-resistant stem cells were sensitized to tyrosine kinase inhibitors by treatment with a pan-Bcl-2 inhibitor.¹⁴ In solid tumors, the role of Bcl-2 family members in regulating the stem cell compartment is less clear. By analyzing the expression and relative function of Bcl-2 and Bcl-X_L in LCSC, we identified a prevalent role of Bcl-X_L in LCSC survival. Differently from chemotherapy, ABT-737 showed a preferential cytotoxic activity towards quiescent/slowly proliferating LCSC in vitro indicating a potential use of this inhibitor to eradicate chemotherapy-resistant LCSC. In vivo, ABT-737 blocked the progression of LCSC-derived xenografts and reduced CSC content, substantiating its specific effect on the CSC compartment. Altogether, these results indicate for the first time a key role of Bcl-X_L in LCSC, opening new perspectives for the elimination of therapy-resistant cells.