Direct and selective pharmacological disruption of the YAP-TEAD interface by IAG933 inhibits Hippo-dependent and RAS-MAPK-altered cancers.

The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.

CRISPR Screening Identifies Mechanisms of Resistance to KRASG12C and SHP2 Inhibitor Combinations in Non-Small Cell Lung Cancer.

Although KRASG12C inhibitors show clinical activity in patients with KRAS G12C mutated non-small cell lung cancer (NSCLC) and other solid tumor malignancies, response is limited by multiple mechanisms of resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155, and the combination is currently under clinical evaluation. To identify rational combination strategies that could help overcome or prevent some types of resistance, we evaluated the duration of tumor responses to JDQ443 ± TNO155, alone or combined with the PI3Kα inhibitor alpelisib and/or the cyclin-dependent kinase 4/6 inhibitor ribociclib, in xenograft models derived from a KRASG12C-mutant NSCLC line and investigated the genetic mechanisms associated with loss of response to combined KRASG12C/SHP2 inhibition. Tumor regression by single-agent JDQ443 at clinically relevant doses lasted on average 2 weeks and was increasingly extended by the double, triple, or quadruple combinations. Growth resumption was accompanied by progressively increased KRAS G12C amplification. Functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles to identify adaptive mechanisms of resistance revealed sensitizing and rescuing genetic interactions with KRASG12C/SHP2 coinhibition; FGFR1 loss was the strongest sensitizer, and PTEN loss the strongest rescuer. Consistently, the antiproliferative activity of KRASG12C/SHP2 inhibition was strongly enhanced by PI3K inhibitors. Overall, KRAS G12C amplification and alterations of the MAPK/PI3K pathway were predominant mechanisms of resistance to combined KRASG12C/SHP2 inhibitors in preclinical settings. The biological nodes identified by CRISPR screening might provide additional starting points for effective combination treatments. SIGNIFICANCE: Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches. See related commentary by Johnson and Haigis, p. 4005.

JDQ443, a Structurally Novel, Pyrazole-Based, Covalent Inhibitor of KRASG12C for the Treatment of Solid Tumors.

Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRASG12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

Genetic heterogeneity and clonal evolution during metastasis in breast cancer patient-derived tumor xenograft models.

Genetic heterogeneity within a tumor arises by clonal evolution, and patients with highly heterogeneous tumors are more likely to be resistant to therapy and have reduced survival. Clonal evolution also occurs when a subset of cells leave the primary tumor to form metastases, which leads to reduced genetic heterogeneity at the metastatic site. Although this process has been observed in human cancer, experimental models which recapitulate this process are lacking. Patient-derived tumor xenografts (PDX) have been shown to recapitulate the patient's original tumor's intra-tumor genetic heterogeneity, as well as its genomics and response to treatment, but whether they can be used to model clonal evolution in the metastatic process is currently unknown. Here, we address this question by following genetic changes in two breast cancer PDX models during metastasis. First, we discovered that mouse stroma can be a confounding factor in assessing intra-tumor heterogeneity by whole exome sequencing, thus we developed a new bioinformatic approach to correct for this. Finally, in a spontaneous, but not experimental (tail-vein) metastasis model we observed a loss of heterogeneity in PDX metastases compared to their orthotopic "primary" tumors, confirming that PDX models can faithfully mimic the clonal evolution process undergone in human patients during metastatic spreading.

SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.

Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments.

FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.

Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Maximizing the Efficacy of MAPK-Targeted Treatment in PTENLOF/BRAFMUT Melanoma through PI3K and IGF1R Inhibition.

The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kß inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kß inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kß, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kß + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response.

Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials.

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.

K-RAS mutant pancreatic tumors show higher sensitivity to MEK than to PI3K inhibition in vivo.

Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.

Identification of elongation factor G as the conserved cellular target of argyrin B.

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

Characterization of the mechanism of action of the pan class I PI3K inhibitor NVP-BKM120 across a broad range of concentrations.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G(2)-M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 µmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells.

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.

p85 Associates with unphosphorylated PTEN and the PTEN-associated complex.

The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110beta isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.

Dual regulatory roles of phosphatidylinositol 3-kinase in IFN signaling.

PI3K is activated by the type I and II IFN receptors, but its precise role in the generation of IFN responses is not well understood. In the present study we used embryonic fibroblasts from mice with targeted disruption of the genes encoding for both the p85alpha and p85beta regulatory subunits of PI3'-kinase (p85alpha(-/-)beta(-/-)) to precisely define the role of PI3K in the control of IFN-induced biological responses. Our data demonstrate that PI3K plays dual regulatory roles in the induction of IFN responses by controlling both IFN-alpha- and IFN-gamma-dependent transcriptional regulation of IFN-sensitive genes and simultaneously regulating the subsequent initiation of mRNA translation for such genes. These processes include the Isg15, Cxcl10, and/or Irf7 genes, whose functions are important in the generation of the biological effects of IFNs. Consistent with this, the induction of IFN antiviral responses is defective in double p85alpha/p85beta knockout cells. Thus, integration of signals via PI3K is a critical event during engagement of the IFN receptors that complements both the transcriptional activity of Jak-STAT pathways and controls initiation of mRNA translation.

Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells.

Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p110alpha yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p110alpha mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110alpha isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition.

Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement.

Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.

Phosphoinositide 3-kinase catalytic subunit deletion and regulatory subunit deletion have opposite effects on insulin sensitivity in mice.

Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85alpha or p85beta exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85alpha-/- p55alpha-/- p50alpha-/- mice, where a decrease in p110alpha and p110beta catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110alpha+/- p110beta+/- mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110alpha+/- p110beta+/- mice showed a approximately 50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.