Inhibitors of oncogenic B-RAFV600E and MKK1/2 have yielded remarkable responses in B-RAF V600E -positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF V600E and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF V600E melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF V600E inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation. Molecular & Cellular

beta-catenin ͉ blast crisis ͉ CML ͉ S17 stromal cells ͉ tyrosine kinase

The success of imatinib mesylate (STI571, Gleevec) in treating chronic myeloid leukemia (CML) is, to date, the crowning achievement of targeted molecular therapy in cancer. Nearly 90% of newly diagnosed patients treated with imatinib in the chronic phase of the disease achieve a complete cytogenetic response. However, more than 95% of these patients retain detectable levels of BCR-ABL mRNA and patients discontinuing imatinib therapy almost invariably relapse, demonstrating that an imatinib insensitive population of leukemia-initiating cells (LICs) persists in nearly all patients. These findings underscore the need for treatments specifically targeting the leukemia-initiating population of CML cells. While mounting evidence suggests that the LIC in the chronic phase of CML is the BCR-ABL positive hematopoietic stem cell, several recent publications suggest that during CML blast crisis, a granulocyte-macrophage progenitor (GMP) population also acquires LIC properties through activation of the β-catenin pathway. Characterization of these cells and evaluation of their sensitivity to imatinib is critical to our understanding and treatment of CML blast crisis.

No abstract

The cyclin-dependent kinase inhibitor p21Cip1 plays an important role in the cellular response to DNA damage. In normal cells, genotoxic stress activates the ATM-p53 pathway that up-regulates the expression of p21 Cip1 leading to cell cycle arrest. However, we have found that in several neoplastic cell lines, ionizing radiation (IR) induces ubiquitin-dependent degradation of p21Cip1 . This process is independent of the ATM pathway as it occurs in immortalized A-T fibroblasts. Knockdown of Skp2, an F-box protein capable of regulating the normal turnover of p21 Cip1 , does not prevent the IR-induced degradation. Instead, this process requires the Cul4-DDB1 Cdt2 E3 ligase as knockdown of either DDB1 or Cdt2 rescues p21Cip1 degradation after IR. Mutating the proliferating cell nuclear antigenbinding site of p21Cip1 also prevents its IR-induced degradation suggesting that the p21 Cip1 -proliferating cell nuclear antigen interaction is critical for this event. Although ectopic expression of a nondegradable p21Cip1 did not by itself affect the clonogenic survival of HEK293 cells after IR, the degradation of p21Cip1 and other targets of the Cul4-DDB1 Cdt2 E3 ligase may collectively contribute to the survival of neoplastic cells after ionizing radiation.