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Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias. Loss of function of the minor spliceosome component ZRSR2 enhances hematopoietic stem cell self-renewal through minor intron retention of its target LZTR1, which is a regulator of RAS-related GTPases. Minor intron retention of LZTR1 was also identified in Noonan syndrome and diverse solid tumor types.

RIT1 is a member of the Ras family of GTPases that direct broad cellular physiological responses through tightly controlled signaling networks. The canonical Ras GTPases are well-defined regulators of the RAF/MEK/ERK pathway and mutations in these are pathogenic in cancer and a class of developmental disorders termed RASopathies. Emerging clinical evidences have now demonstrated a role for RIT1 in RASopathies, namely Noonan syndrome, and various cancers including lung adenocarcinoma and myeloid malignancies. While RIT1 has been mostly described in the context of neuronal differentiation and survival, the mechanisms underlying aberrant RIT1-mediated signaling remain elusive. Here, we will review efforts undertaken to characterize the biochemical and functional properties of the RIT1 GTPase at the molecular, cellular, and organismal level, as well as provide a phenotypic overview of different human conditions caused by RIT1 mutations. Deeper understanding of RIT1 biological function and insight to its pathogenic mechanisms are imperative to developing effective therapeutic interventions for patients with RIT1-mutant Noonan syndrome and cancer.

The spindle assembly checkpoint (SAC) functions as a sensor of unattached kinetochores that delays mitotic progression into anaphase until proper chromosome segregation is guaranteed.(1,2) Disruptions to this safety mechanism lead to genomic instability and aneuploidy, which serve as the genetic cause of embryonic demise, congenital birth defects, intellectual disability, and cancer.(3,4) However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated and this process is prominently regulated by the Ras family of small guanosine triphosphatases (GTPases).(5) Here we show that RIT1, a Ras-related GTPase that regulates cell survival and stress response,(6) is essential for timely progression through mitosis and proper chromosome segregation. RIT1 dissociates from the plasma membrane (PM) during mitosis and interacts directly with SAC proteins MAD2 and p31(comet) in a process that is regulated by cyclin-dependent kinase 1 (CDK1) activity. Furthermore, pathogenic levels of RIT1 silence the SAC and accelerate transit through mitosis by sequestering MAD2 from the mitotic checkpoint complex (MCC). Moreover, SAC suppression by pathogenic RIT1 promotes chromosome segregation errors and aneuploidy. Our results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism.

Human oncoproteins promote transformation of cells into tumours by dysregulating the signalling pathways that are involved in cell growth, proliferation and death. Although oncoproteins were discovered many years ago and have been widely studied in the context of cancer, the recent use of high-throughput sequencing techniques has led to the identification of cancer-associated mutations in other conditions, including many congenital disorders. These syndromes offer an opportunity to study oncoprotein signalling and its biology in the absence of additional driver or passenger mutations, as a result of their monogenic nature. Moreover, their expression in multiple tissue lineages provides insight into the biology of the proto-oncoprotein at the physiological level, in both transformed and unaffected tissues. Given the recent paradigm shift in regard to how oncoproteins promote transformation, we review the fundamentals of genetics, signalling and pathogenesis underlying oncoprotein duality.

Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells. Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D). In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients. Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L). While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. Figure 1 Figure 1. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Mutations in RNA splicing factors are amongst the most common genetic alterations in myeloid malignancies. Mutations in the splicing factors SF3B1, SRSF2, and U2AF1 occur as heterozygous, missense mutations and have been shown to confer a change-of-function. In contrast, the X chromosome encoded ZRSR2 is enriched in nonsense/frameshift mutations in males, consistent with loss of function. To date however, we do not understand the basis for enrichment of ZRSR2 mutations in leukemia. Moreover, ZRSR2 is the only one of these factors that primarily functions in the minor spliceosome. While most introns are spliced by the major spliceosome, a small subset ( The rarity and conservation of minor introns offered a unique opportunity to investigate splicing factor mutations and identify potential tissue-specific roles of the minor spliceosome. Modeling loss-of-function mutations in ZRSR2 via a mouse model for induced deletion of Zrsr2 revealed strikingly enhanced self-renewal of Zrsr2-deficient male and female hematopoietic cells (Fig. A-C). This was in stark contrast to the effects of hotspot mutations in Sf3b1and Srsf2 and similar to those of Tet2 loss on increasing self-renewal and numbers of HSCs. Zrsr2 loss was also associated increased myeloid cells in the blood and long-term hematopoietic stem cells (HSCs) in the marrow (Fig. C). To understand the mechanistic basis by which ZRSR2 loss causes aberrant HSC self-renewal, we quantified transcriptome-wide splicing patterns in MDS patients. ZRSR2-mutant samples had widespread, dysfunctional recognition of minor introns- 48% of minor introns exhibiting significantly increased retention (Fig. D). We next systematically mimicked the effects of nonsense-mediated decay caused by minor intron retention in ZRSR2-mutants. Every gene containing a ZRSR2-regulated minor intron was targeted by 4 sgRNAs via a positive-enrichment CRISPR screen using pools of lentiviral sgRNAs in cytokine-dependent human and mouse hematopoietic cell lines. This identified several minor intron-containing genes whose downregulation conferred cytokine independence. Strikingly, just one gene was enriched in all lines (Fig. E): LZTR1, a cullin-3 adaptor for ubiquitin-mediated suppression of RAS-related GTPases which is subject to loss-of-function mutations in several cancers and the RASopathy Noonan Syndrome. Minor intron retention in LZTR1 correlated with reduced LZTR1 protein in MDS patients (Fig. F-G). Inducing mutations in either the protein-coding region of LZTR1 or its minor intron resulted in cytokine independence (Fig. H), reduced LZTR1, and dramatic accumulation of RIT1, a RAS GTPase substrate of LZTR1. In a Noonan Syndrome family wherein one child died of AML, the mother and all children carried an intronic mutation within LZTR1's minor intron (Fig. I-J). Fibroblasts from each family member revealed clear LZTR1 minor intron retention with impaired LZTR1 protein expression and RIT1 accumulation in subjects bearing the LZTR1 minor intron mutation (Fig. J). We next interrogated LZTR1 minor intron splicing across all cancers in the TCGA. While LZTR1's minor intron was efficiently excised in normal samples, a notable subset of tumors in almost all cancer types exhibited significantly increased retention within LZTR1's minor intron. These data indicate LZTR1 is frequently dysregulated via perturbed minor intron splicing - much more so than by protein-coding mutations alone. Here we uncover a heretofore unrecognized role of minor intron excision in regulating HSC self-renewal, a molecular link between ZRSR2 mutations and aberrant LZTR1 splicing and expression, and frequent LZTR1 minor intron retention in diverse cancers and cancer predisposition syndromes. Given frequent post-transcriptional disruption of LZTR1 in the absence of protein-coding mutations, our data additionally motivate study of other cancer-associated minor intron-containing genes which may be dysregulated via similar, and as-yet-undetected, aberrant splicing. Figure Disclosures Abdel-Wahab: Merck: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy.

Defective degradation as disease mechanism Ubiquitination often targets proteins for destruction. Castel et al. describe a mechanism by which mutations in the small guanine triphosphatase RIT1 may act to cause certain developmental disorders and cancers. They detected a protein, LZTR1, that interacted with wild-type RIT1 but not with oncogenic mutant forms of RIT1. LZTR1 acts as a substrate-specific adaptor for a ubiquitin ligase. Altered forms of RIT1 that are not subject to ubiquitin-mediated degradation thus accumulate. Because RIT1 functions in growth factor signaling and excessive signaling, these findings may explain the malignancies associated with RIT1 mutations. Science , this issue p. 1226

PIK3CA (which encodes the PI3K alpha isoform) is the most frequently mutated oncogene in breast cancer. Small-molecule PI3K inhibitors have shown promise in clinical trials; however, intrinsic and acquired resistance limits their utility. We used a systematic gain-of-function approach to identify genes whose upregulation confers resistance to the PI3K inhibitor BYL719 in breast cancer cells. Among the validated resistance genes, Proviral Insertion site in Murine leukemia virus (PIM) kinases conferred resistance by maintaining downstream PI3K effector activation in an AKT-independent manner. Concurrent pharmacologic inhibition of PIM and PI3K overcame this resistance mechanism. We also observed increased PIM expression and activity in a subset of breast cancer biopsies with clinical resistance to PI3K inhibitors. PIM1 overexpression was mutually exclusive with PIK3CA mutation in treatment-naïve breast cancers, suggesting downstream functional redundancy. Together, these results offer new insights into resistance to PI3K inhibitors and support clinical studies of combined PIM/PI3K inhibition in a subset of PIK3CA-mutant cancers. Significance: PIM kinase overexpression confers resistance to small-molecule PI3K inhibitors. Combined inhibition of PIM and PI3K may therefore be warranted in a subset of breast cancers. Cancer Discov; 6(10); 1134–47. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 1069

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2. Heterogeneous 89Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2-amplified EG cancer. Significance: Analysis of patients with ERBB2-amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR/ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones. See related commentary by Klempner and Catenacci, p. 166. This article is highlighted in the In This Issue feature, p. 151

Estrogen Receptor (ER) and the phosphoinositide 3-kinase (PI3K) pathways participate in regulatory crosstalk in breast cancer. We identified that chromatin regulation is at the intersection of oncogenic PI3K and ER. The PI3K effectors AKT, also known as protein kinase B (PKB), and SGK (serum/glucocorticoid-regulated kinase) play a redundant role by phosphorylating the chromatin regulator KMT2D and modulating ER activity and therapy resistance.

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT‐474 breast cancer cell‐derived tumours in a xenograft model. Finally, we present the kinome‐wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34‐SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.

Activating mutations in PIK3CA, the gene encoding phosphoinositide-(3)-kinase α (PI3Kα), are frequently found in estrogen receptor (ER)–positive breast cancer. PI3Kα inhibitors, now in late-stage clinical development, elicit a robust compensatory increase in ER-dependent transcription that limits therapeutic efficacy. We investigated the chromatin-based mechanisms leading to the activation of ER upon PI3Kα inhibition. We found that PI3Kα inhibition mediates an open chromatin state at the ER target loci in breast cancer models and clinical samples. KMT2D, a histone H3 lysine 4 methyltransferase, is required for FOXA1, PBX1, and ER recruitment and activation. AKT binds and phosphorylates KMT2D, attenuating methyltransferase activity and ER function, whereas PI3Kα inhibition enhances KMT2D activity. These findings uncover a mechanism that controls the activation of ER by the posttranslational modification of epigenetic regulators, providing a rationale for epigenetic therapy in ER-positive breast cancer.

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

Epithelial morphogenesis and stability are essential for normal development and organ homeostasis. The mouse neural plate is a cuboidal epithelium that remodels into a columnar pseudostratified epithelium over the course of 24 hr. Here we show that the transition to a columnar epithelium fails in mutant embryos that lack the tumor suppressor PTEN, although proliferation, patterning and apical-basal polarity markers are normal in the mutants. The Pten phenotype is mimicked by constitutive activation of PI3 kinase and is rescued by the removal of PDK1 (PDPK1), but does not depend on the downstream kinases AKT and mTORC1. High resolution imaging shows that PTEN is required for stabilization of planar cell packing in the neural plate and for the formation of stable apical-basal microtubule arrays. The data suggest that appropriate levels of membrane-associated PDPK1 are required for stabilization of apical junctions, which promotes cell elongation, during epithelial morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.12034.001, eLife digest In mammals, the brain and spinal cord develop from a flat sheet of cells called the neural plate, which bends around to create a structure known as the neural tube. This bending process occurs through a complex sequence of cell shape changes. The cells in the neural plate are initially short and wide, but transform into long, thin cells as the neural plate forms. Problems that prevent the neural tube from forming correctly are amongst the most common birth defects in humans. Many cancer cells contain a mutation that affects a gene that produces a protein called PTEN. This protein normally activates a tumor suppressor pathway, and so cancer cells that lack PTEN divide and grow uncontrollably. Grego-Bessa et al. have now examined mouse embryos that lack this gene, and found that the neural plate in such embryos forms irregular ruffles rather than a closed tube. Further investigation revealed that the neural tube defects are not due to the inactivation of the traditional tumor suppressor pathway. Instead, correct neural tube formation relies upon the ability of PTEN to remove phosphate groups from a target lipid, which is important for limiting the activity of an enzyme called PDK1. Unlimited PDK1 activity causes complex changes that prevent the neural plate cells from elongating and packing together correctly. Future work is now needed to investigate the exact molecules targeted by PDK1 and the roles they play in disorders and diseases caused by a lack of the PTEN protein. DOI: http://dx.doi.org/10.7554/eLife.12034.002

Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic RTK signaling leads to phosphorylation of Spred1(S105) in the EVH domain, which disrupts Spred1-NF1 binding and function. Phosphomimetic Spred1 is unable to suppress Ras-GTP following growth factor stimulation and cancer cell proliferation. The Spred1(S105) kinase is likely to be a CDK based on in vitro kinase and in vivo cell line assays. Our findings provide one potential mechanism by which oncogenic RTK signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Pau Castel, Jillian Silva, Jacqueline Galeas, Kathy Li, Anatoly Urisman, Frank McCormick. Oncogenic RTK signaling inhibits Spred1/NF1 to sustain constitutive Ras/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5520.

Summary PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells., Graphical Abstract, Highlights • The PDK1-SGK1 axis can sustain mTORC1 activity upon PI3K/AKT suppression • SGK1 directly phosphorylates TSC2, resulting in mTORC1 activation • Targeting PDK1 sensitizes PIK3CA-mutant cells to PI3Kα inhibition, Castel et al. show that PDK1 activates SGK1 to phosphorylate and inhibit TSC2, contributing to maintenance of mTORC1 activity and resistance of PIK3CA-mutant cancer cells to PI3Kα inhibition. Targeting either PDK1 or SGK1 restores the sensitivity of these resistant cancer cells to PI3Kα inhibition.

Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Somatic mutations in PIK3CA, the gene encoding the α isoform of PI3K (p110α), are frequent in breast cancer. These mutations lead to constitutive activation of the PI3K/AKT/mTORC1 pathway and are a potential therapeutic target. Despite the encouraging activity shown by selective PI3Kα inhibitors in early clinical studies, many breast cancer patients with PIK3CA-mutated tumors remain insensitive to these agents. We have previously shown both in preclinical models and in patients undergoing treatment with PI3Kα inhibitors that intrinsic resistance to PI3Kα inhibition is associated with sustained mTORC1 activation despite full AKT blockade. To elucidate how tumor cells can activate mTORC1 bypassing the AKT node, we performed a synthetic lethal RNAi screening using a library against the human kinome and phosphatome. We found that knockdown of phosphoinositide-dependent kinase 1 (PDK1) sensitized resistant cell lines to PI3Kα blockade, preventing mTORC1 signaling. These results were also validated by either shRNA-mediated PDK1 genetic knockdown or using the selective PDK1 kinase inhibitor, GSK2334470. The combination with PI3Kα inhibition abolished mTORC1 activity and resulted in superior antitumor activity in vitro and in vivo. Mechanistically, the combination of GSK2334470 with PI3Kα inhibition decreased the phosphorylation of the transcription factor FOXO3 at its T32 residue, causing its nuclear translocation, occupancy at the promoters of its target genes, and increased transcription. Given that full suppression of AKT activity was already achieved with PI3Kα inhibition alone, we hypothesized that another kinase downstream of PDK1 is responsible for FOXO3 phosphorylation and is inhibited upon PDK1 blockade. We demonstrated that SGK1, an AGC kinase upstream of FOXO3 and downstream of PDK1, is responsible for mediating resistance to PI3Kα inhibitors by activating mTORC1 through the direct phosphorylation of the tumor suppressor TSC2. Consistently, we found that SGK1 mRNA and protein levels are higher in PIK3CA-mutant cells resistant to PI3Kα. Our results were further corroborated through the characterization of the biochemical properties of a first-in-class SGK1 kinase inhibitor. This compound, in combination with a PI3Kα inhibitor, phenocopied the effects of PDK1 inhibition in all the models tested. Our findings uncover a parallel signaling cascade triggered by PI3Kα pharmacological inhibition that reconciles the sustained mTORC1 activation with lack of AKT enzymatic activity. This offers a novel therapeutic approach for PIK3CA-mutant breast tumors intrinsically resistant to PI3Kα inhibition. Citation Format: Pau Castel, Haley Ellis, Ruzica Bago, Eneda Toska, Kannan Srinivasaraghavan, F Javier Carmona, Pedram Razavi, Chandra Verma, Maura Dickler, Sarat Chandarlapaty, Edi Brogi, Dario Alessi, José Baselga, Maurizio Scaltriti. PDK1 blockade overcomes intrinsic resistance to PI3Kα inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2107.

Activating mutations of PIK3CA, the gene encoding the p110α subunit of PI3K, are frequent in breast cancer and selective inhibitors of this enzyme have shown promising clinical activity in breast tumors harboring these mutations. We studied the case of a patient with metastatic breast cancer harboring a PIK3CA mutation that was treated in a clinical trial with BYL719, a highly selective PI3Kα inhibitor. The treatment resulted in a robust partial response that lasted 7 months followed by rapid progression and death. A rapid autopsy was performed with collection of tissue samples from 16 different metastatic sites. We compared by whole genome and exome sequencing the original primary tumor, a rapidly progressing lung metastasis and a periaortic lesion that was still responding to BYL719 at time of death. Besides several common alterations, PTEN loss and a missense mutation were detected only in the lung metastasis. Using targeted exome sequencing we analyzed all the other available samples. Strikingly, we observed a consistent loss in PTEN (via different mechanisms such as deletion, splice site mutation and frameshift mutations) in all the lesions refractory to BYL719 but not in the responding ones. In every case, the loss of PTEN was also documented by lack of protein expression by immunohistochemistry (IHC). Finally, we were able to build a dendrogram showing the phylogenetic evolution of the lesions and the evolutionary convergence of the PTEN alterations. To validate PTEN loss as a possible mechanism of acquired resistance to selective PI3Kα inhibition, we generated doxycycline-inducible PTEN shRNA stable clones starting from three different BYL719-sensitive cell lines. In all the studied models, induction of PTEN shRNA resulted in resistance to BYL719. Since PTEN deficient genetic models have been shown to rely on the β subunit of the PI3K holoenzyme, we tested whether the concomitant inhibition of both p110α and p110β was sufficient to revert the resistant phenotype. BKM120 (a pan-PI3K inhibitor) or the addition of AZD6482 (p110β inhibitor) to BYL719 re-sensitized the cells to BYL719. To expand our findings in vivo, we generated a patient-derived xenograft (PDX) model from a PTEN-null non-responding lesion (lung). Consistently, this PDX model was refractory to the antitumor activity of BYL719 but conserved sensitivity to BKM120 or the combination of AZD6482 and BYL719. In both cases, IHC analysis revealed a decrease in PI3K/AKT downstream effectors pPRAS40 (246) and pS6 (240/4) staining with BKM120 or AZD6482+BYL719, but not with BYL719 alone. Preliminary analyses of other samples collected from patients treated with BYL719 suggest that PTEN loss is a relatively frequent event upon therapy progression. Taken together, the different mechanisms that inactivate PTEN in the tumor treated with BYL719 can be explained by convergent phenotypic evolution in a heterogeneous tumor and highlight the importance of PTEN and PI3Kβ in acquired resistance to PI3Kα inhibitors. Citation Format: Pau Castel, Dejan Juric, Helen Won, Benjamin Ainscough, Haley Ellis, Saya Ebbesen, Malachi Griffith, Obi Griffith, Iyer Gopakumar, Dennis Sgroi, Steven Isakoff, Elaine Mardis, David Solit, Scott Lowe, Cornelia Quadt, Malte Peters, Michael Berger, Maurizio Scaltriti, José Baselga. Loss of PTEN leads to clinical resistance to the PI3Kα inhibitor BYL719 and provides evidence of convergent evolution under selective therapeutic pressure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2014-LB-327

Triple negative breast cancer (TNBC) is clinically defined by lack of expression of estrogen and progesterone receptors and no overexpression/amplification of human epidermal growth factor receptor 2 (HER2). It affects approximately one-fourth of breast cancer patients and is usually characterized by an aggressive phenotype with high mitotic index and poor prognosis. No TNBC-targeted therapeutic exists and the standard of care is currently limited to cytotoxic chemotherapy. Overexpression of human epidermal growth factor receptor (EGFR) as well as inactivation or decreased expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are frequent in TNBC. Thus, inhibition of EGFR and/or the PI3K/Akt pathway are attractive therapeutic strategies for this malignancy. Preclinical evidence, however, suggests that inhibition of the PI3K/Akt axis induces compensatory expression and activation of upstream receptor tyrosine kinases, including EGFR and, most prominently, human epidermal growth factor receptor 3 (HER3). This may reduce the antitumor effects of single-agent PI3K pathway blockade. Furthermore, studies using in vitro models of resistance to the anti-EGFR antibody cetuximab suggest that HER3 expression itself can limit the sensitivity to this agent by increasing EGFR/HER3 heterodimerization and activation of downstream pathways. In this work we hypothesized that targeting both EGFR and HER3, in combination with PI3K/Akt inhibition, would result in enhanced therapeutic activity in EGFR-positive TNBC. We treated PTEN-negative EGFR-positive HCC70 and MDA-MB-468 TNBC cell lines with GDC-0068, a selective inhibitor of the Akt 1/2/3 isoforms, GDC-0941, a class I selective pan-PI3K inhibitor, MEHD7945A, an antibody targeting both EGFR and HER3 and the combinations of these inhibitors. Compared to single agents, the combination of GDC-0068 or GDC-0941 and MEHD7945A prevented the induction of EGFR and HER3 phosphorylation, enhanced the inhibition of the PI3K and ERK downstream pathways and resulted in superior inhibition of cell proliferation/viability compared to single agents. To expand our findings in vivo, we first tested the efficacy of MEHD7945A in combination with either the Akt or PI3K inhibitor in both MDA-MB-468- and HCC70-derived xenografts. While the tumors responded only modestly to single agent GDC-0068, GDC-0941 and MEHD7945A, the combination of GDC-0068 or GDC-0941 with MEHD7945A yielded superior tumor growth inhibition compared to monotherapies. We next investigated the levels of EGFR and HER3 expression/activation in HCC70 tumors collected at the end of the experiments by reverse-phase protein detection. As expected, increase of both EGFR and HER3 expression and phosphorylation induced by Akt or PI3K was prevented by the addition of MEHD7945A. We then tested the activity of the same treatments in a patient-derived xenograft (PDX) model of TNBC available in our laboratory. These tumors were characterized by undetectable levels of PTEN, high levels of EGFR and ∼70% staining for Ki67. When used as single agents, GDC-0068, GDC-0941 and MEHD7945A delayed tumor growth. However, their combinations elicited durable tumor stasis. Consistently with the cell-based xenografts, MEHD7945A counteracted EGFR and HER3 phosphorylation caused by PI3K/Akt inhibition. Collectively, these data show that targeting both EGFR and HER3 enhances the antitumor effects of PI3K/Akt inhibitors. In order to dissect the role of HER3 inhibition in these models, we compared the activity of cetuximab with MEHD7945A in combination with either GDC-0068 or GDC-0941 in HCC70 cells. Both antibodies enhanced the antiproliferative activity mediated by PI3K/Akt inhibition in cells stimulated with the EGFR ligand epidermal growth factor; however MEHD7945A was superior to cetuximab in cooperating with the antiproliferative activity of GDC-0068 and GDC-0941 in cells stimulated with the HER3 ligand heregulin. We next compared the antitumor activity of cetuximab and MEHD7945A in combination with GDC-0941 in HCC70-derived xenografts. While concomitant targeting of EGFR, HER3 and PI3K was superior to monotherapies and led to tumor shrinkage, the combination of cetuximab and GDC-0941 did not result to any further inhibition of tumor growth compared to single agent treatments. Biochemically, both MEHD7945A and cetuximab blocked EGFR phosphorylation in these xenografts; however, only MEHD7945A decreased HER3 activation. These results confirm that HER3 plays an important role in limiting the efficacy of PI3K inhibition in this setting. To investigate whether changes in EGFR and HER3 levels can affect the response to anti-EGFR therapy in TNBC patients, we measured the expression of these receptors in samples from patients enrolled in two pilot neoadjuvant clinical trials testing the antitumor activity of the anti-EGFR antibodies panitumumab (40 patients) and cetuximab (30 patients) in combination with standard chemotherapy. Of 40 patients treated with panitumumab-based therapy 19 (47.5%) achieved pathological complete response (pCR) after 24 weeks and 21 (52.5%) showed residual disease at the time of surgery. We measured EGFR and HER3 expression by immunohistochemistry in the 40 pre-treatment specimens and in the 21 residual invasive tumors excised at surgery. Patients with higher baseline EGFR expression were more likely to achieve pCR. Moreover, EGFR levels were decreased in the residual tumors of nine out of 21 non-pCR patients when compared to the paired baseline specimens (p=0.07). HER3 staining, available for 13 non-responder patients, showed higher HER3 expression in the residual lesions of 7 out of 13 non-pCR patients compared to the paired pre-treatment samples (p=0.01). Of 30 patients enrolled in the study testing the antitumor activity of cetuximab-based therapy, 9 experienced pCR. Consistently, HER3 expression was found upregulated in the residual tumors of 11 out of 19 non-pCR patients when compared to the baseline paired specimens (p=0.103). These results suggest that high EGFR expression may be required for optimal response to anti-EGFR therapeutic antibodies and that HER3 expression increases following anti-EGFR therapy in patients that do not experience complete tumor regression. In this work we provide evidence that HER3 plays a potential role in limiting the antitumor activity of both PI3K/Akt inhibitors and anti-EGFR agents. We demonstrate that simultaneous targeting of EGFR and HER3 is required to enhance the efficacy of PI3K/Akt inhibition in preclinical models of EGFR-positive TNBC. Further, our clinical findings suggest that HER3 expression is induced in TNBC patients with a lower probability of achieving tumor remission upon anti-EGFR therapy. Therefore, the rationale behind targeting simultaneously EGFR, HER3 and PI3K in TNBC is based on: 1) nearly half of TNBCs express high levels of EGFR, 2) TNBCs often express low levels of PTEN and 3) HER3 upregulation may limit their sensitivity to targeted therapy. In addition, tumor cells that express relatively high levels of these receptors can function as molecular “flags” for immune-mediated antibody dependent cytotoxicity, intrinsic characteristics of IgG type I antibodies such as MEHD7945A in vivo. Given that both pharmacokinetic and pharmacodynamic data for MEHD7945A, GDC-0068 and GDC-0941 are already available, the design of phase II clinical trials testing the activity of these possible combinations in TNBC would be straightforward. Patient enrollment may be based on high EGFR expression with the requirement of mandatory “on-treatment” biopsies to evaluate both pathway inhibition and possible HER3 upregulation upon therapy. In conclusion, we believe that simultaneous inhibition of EGFR, HER3 and the PI3K/Akt pathway has the potential to greatly expand the percentage of TNBC patients who can benefit from targeted therapy. Citation Format: Maurizio Scaltriti, Jessica Tao, Pau Castel, Nina Radosevic-Robin, Moshe Elkabets, Neil Auricchio, Dejan Juric, Frédérique Penault-Llorca, Jose Baselga. Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG01. doi:10.1158/1538-7445.AM2014-NG01

The PI3K/AKT/mTOR signaling is important for cell proliferation, survival, and metabolism. Hyperactivation of this pathway is one of the most common signaling abnormalities observed in cancer and a substantial effort has recently been made to develop molecules targeting this signaling cascade. However, it is becoming evident that PI3K inhibitors used as single agents do not elicit dramatic or durable responses. Given the numerous mechanisms mediating intrinsic and acquired resistance to these agents, hypothesis-based combinatorial strategies are probably needed to fully exploit their antitumor activity. In the first part of this review, we briefly dissect the PI3K/AKT/mTOR axis and list the most advanced compounds targeting different nodes of this cascade. The second part focuses on what we believe to be the most promising rationale-based therapeutic combinations with PI3K/AKT/mTOR inhibitors in solid tumors, with special emphasis on breast cancer.

Background: Up to 60% of triple negative breast cancers (TNBCs) express high levels of EGFR. Moreover, TNBCs are associated with increased frequency of phosphatase and tension homologue (PTEN) loss of function, leading to hyperactivation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. This provides the rationale for using PI3K/AKT inhibitors in this subset of patients. However, compensatory expression of receptor tyrosine kinases (RTKs) such as HER3 may limit efficacy of PI3K/AKT inhibitors. Therefore, we hypothesize that combined targeting of both EGFR and HER3 and the PI3K/AKT pathway will result in superior antitumor activity compared to single agent in TNBC. Materials and Methods: Several TNBC cell lines were treated with MEHD7945A, a dual-action antibody that targets both EGFR and HER3, AKT inhibitor GDC-0068, and pan-PI3K inhibitor GDC-0941. Cell viability was measured by CellTiter-Glo and Crystal Violet. Both cell line- and patient-derived xenograft models of TNBC were treated with MEHD7945A, GDC-0068, GDC-0941, or the combination of MEHD7945A with either GDC-0068 or GDC-0941. Tumor size and histology were examined. Protein expression was measured by Western blot, Mass Spectometry, CEER and immunohistochemistry. Results: GDC-0068 and GDC-0941 treatment resulted in variable inhibition of cell viability, with IC50s ranging from 170 nM to >1 μM across all TNBC cell lines. In cells stimulated with either EGF or Heregulin MEHD7945A prevented EGFR/HER3 receptors phosphorylation and improved the antiproliferative activity of the PI3K/AKT inhibitors. To test the activity of these compounds in vivo we used three different models of TNBC, two cell line (MDA-MB-468 and HCC70)-based and a patient-derived xenograft. MEHD7945, GDC-0941 and GDC-0068 showed variable delay in tumor growth whereas combination of MEHD7945A with either GDC-0068 or GDC-0941 was always superior to single agent treatment. Both combinations either prevented tumor growth or led to tumor shrinkage with complete responses achieved in 1/2 mice in each cohorts. Of note, all the treatments (up to 9 weeks of therapeutic exposure) were well tolerated. Analysis of treated tumors reveals potent inhibition of the PI3K/AKT pathway, with decreased levels phospho-PRAS40 and phospho-S6. Moreover, we confirmed that MEHD7945A effectively prevented EGFR and HER3 phosphorylation consequent to PI3K inhibition. Conclusions: Combined therapy with MEHD7945A and either GDC-0068 or GDC-0941 was superior to monotherapy in preclinical models of TNBC. We are currently exploring the levels of expression/activation of EGFR and HER3 following PI3K/Akt blockade in samples from patients treated with GDC-0068. These findings provide the rationale for combined targeting of PI3K/AKT and EGFR/HER3 in triple negative breast cancers. Citation Format: Maurizio Scaltriti, Jessica Tao, Dejan Juric, Neil Auricchio, Pau Castel, Natasha Morse, Phillip Kim, Sharat Singh, Saswati Hazra, Todd Hembrough, Jon Burrows, Jose Baselga. Combined blockade of PI3K/AKT and EGFR/HER3 enhances antitumor activity in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4471. doi:10.1158/1538-7445.AM2013-4471