Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer

© 2016 The Author(s)Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy

Pp2a/b55a substrate recruitment as defined by the retinoblastoma-related protein p107

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55a, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55a recruits p107, a pRB-related tumor suppressor and B55a substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an “HxRVxxV619-625” short linear motif (SLiM) in p107 as necessary for B55a binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55a/PP2A substrates, including TAU, contain a related SLiM C- terminal from a proximal phosphosite, “p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R”. Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55a, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55a groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55a mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55a role in multiple cellular processes. © 2021, eLife Sciences Publications Ltd. All rights reserved.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer

© 2016 The Author(s)Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer

© 2016 The Author(s)Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy

PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55α/PP2A reconstitution that triggers centrosome destabilization.

The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (\u3e75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity

PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55 alpha/PP2A reconstitution that triggers centrosome destabilization

The PPP2R2A gene encodes the B55 alpha regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in similar to 42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55 alpha is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55 alpha reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55 alpha-low cells also appear addicted to lower B55 alpha expression, as even moderate increases in B55 alpha expression are toxic. Reconstitution of B55 alpha expression in prostate cancer (PCa) cell lines with low B55 alpha expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55 alpha reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55 alpha/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55 alpha. Thus, we propose the reduction in B55 alpha levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55 alpha reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55 alpha activity