Benjamin G. Neel

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We quantified the proteomes of 76 human BC cell lines using data independent acquisition (DIA) based proteomics, identifying 6091 proteins. We then established a 24-protein panel distinguishing TNBC from other BC types. Integrating prior multi-omics datasets with the present proteomic results to predict the sensitivity of 90 drugs, we found that proteomics data improved drug sensitivity predictions. The sensitivity of the 90 drugs was mainly associated with cell cytoskeleton, signal transduction and mitochondrial function. We next profiled the proteome changes of nine cell lines (five TNBC cell lines, four non-TNBC cell lines) perturbated by EGFR/AKT/mTOR inhibitors. In the TNBC cell lines, metabolism pathways were dysregulated after EGFR/mTOR inhibitors treatment, while RNA modification and cell cycle pathways were dysregulated after AKT inhibitor treatment. Our study presents a systematic multi-omics and in-depth analysis of the proteome of BC cells. This work aims to aid in prioritization of potential therapeutic targets for TNBC as well as to provide insight into adaptive drug resistance in TNBC.

The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) both considered candidates. Here, by using genetically engineered mouse models and organoids, we assessed the tumor-forming properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation and Tp53 mutation in Pax8 + FTE caused Serous Tubal Intraepithelial Carcinoma (STIC), which metastasized rapidly to the ovarian surface. These events were recapitulated by orthotopic injection of mutant FTE organoids. Engineering the same genetic lesions into Lgr5 + OSE or OSE-derived organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. FTE- and OSE-derived tumors had distinct transcriptomes, and comparative transcriptomics and genomics suggest that human HGSOC arises from both cell types. Finally, FTE- and OSE-derived organoids exhibited differential chemosensitivity. Our results comport with a dualistic origin for HGSOC and suggest that the cell-of-origin might influence therapeutic response.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH−/− PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed “q-oxPTPome,” a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10×. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTPs were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathologic PTP oxidation and neoplastic disease. Significance: This work identifies a novel mechanism of activation of the oncogenic kinase ABL1 via ROS-induced, oxidation-mediated inactivation of cognate protein tyrosine phosphatases.

The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) each suggested as candidates. Here, by using genetically engineered mouse models and novel organoid systems, we assessed the tumor-forming capacity and properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation (via T121 expression) and Tp53 mutation in Pax8+ FTE caused transformation to Serous Tubal Intraepithelial Carcinoma (STIC), which rapidly metastasized to the ovarian surface. This mouse model was recapitulated by FTE organoids, which, upon orthotopic injection, generated widely metastatic HGSOC. The same genetic lesions in Lgr5+ OSE cells or organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. Comparative transcriptome analysis was consistent with different human HGSOCs arising from FTE and OSE. Furthermore, FTE- and OSE-derived organoids showed differential sensitivity to HGSOC chemotherapeutics. Our results comport with a dualistic origin for HGSOC and suggest the cell-of-origin could influence therapeutic response. SIGNIFICANCE: The cell-of-origin for high grade serous ovarian carcinoma (HGSOC) has been controversial. By generating novel GEMMs and organoid models with the same oncogenic defects, we demonstrate that HGSOC can originate from either fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). Importantly, FTE- and OSE-derived tumors differ significantly in biologic properties.

Adaptive resistance to MEK inhibitors (MEKi) typically occurs via induction of genes for different receptor tyrosine kinases (RTK) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 (PTPN11) is required for RAS/ERK pathway activation by most RTKs and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEKi inhibited the proliferation of multiple cancer cell lines in vitro. PTPN11 knockdown/MEKi treatment had similar effects, whereas expressing SHP099 binding–defective PTPN11 mutants conferred resistance, demonstrating that SHP099 is on-target. SHP099/trametinib was highly efficacious in xenograft and/or genetically engineered models of KRAS-mutant pancreas, lung, and ovarian cancers and in wild-type RAS-expressing triple-negative breast cancer. SHP099 inhibited activation of KRAS mutants with residual GTPase activity, impeded SOS/RAS/MEK/ERK1/2 reactivation in response to MEKi, and blocked ERK1/2-dependent transcriptional programs. We conclude that SHP099/MEKi combinations could have therapeutic utility in multiple malignancies. Significance: MEK inhibitors show limited efficacy as single agents, in part because of the rapid development of adaptive resistance. We find that SHP2/MEK inhibitor combinations prevent adaptive resistance in multiple cancer models expressing mutant and wild-type KRAS. Cancer Discov; 8(10); 1237–49. ©2018 AACR. See related commentary by Torres-Ayuso and Brognard, p. 1210. This article is highlighted in the In This Issue feature, p. 1195

Due to the involvement of SHP2 (SH2 domain‐containing protein‐tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC‐87877 does not exhibit robust inhibitory effects on growth factor‐dependent MAPK (mitogen‐activated protein kinase) pathway activation and that the recently developed active site‐targeting SHP2 inhibitors IIB‐08, 11a‐1, and GS‐493 show off‐target effects on ligand‐evoked activation/trans‐phosphorylation of the PDGFRβ (platelet‐derived growth factor receptor β). GS‐493 also inhibits purified human PDGFRβ and SRC in vitro, whereas PDGFRβ inhibition by IIB‐08 and 11a‐1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.

Adaptive resistance to MEK inhibitors (MEK-Is) typically occurs via induction of genes for different receptor tyrosine kinases (RTKs) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 (PTPN11) is required for RAS/ERK pathway activation by most RTKs, and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEK-I inhibits proliferation of multiple cancer cells in vitro. PTPN11 knockdown/MEK-I had similar effects, while expressing SHP099-binding mutants conferred resistance, demonstrating that SHP099 was on-target. This combination was efficacious in xenograft and/or genetically engineered models of KRAS-mutant pancreas cancer and ovarian cancer and in wild-type RAS-expressing triple negative breast cancer. Biochemical studies show that SHP099 impedes SOS/RAS/MEK/ERK1/2 reactivation in response to MEK-Is and blocks ERK1/2-dependent transcriptional programs. SHP099 alone also inhibited RAS activation in some, but not all, KRAS-mutant lines. Hence, SHP099/MEK-I combinations could have therapeutic utility in multiple malignancies.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germ line inactivating alterations in Fumarate Hydratase ( FH ) cause HLRCC, and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH -/- PRCC cells have increased ABL1 activation, which promotes tumor growth, but how ABL1 is activated remained unclear. Oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity; conceivably, ROS-catalyzed inactivation of an ABL-directed PTP might account for ABL1 activation in this malignancy. Previously, our group developed “q-oxPTPome,” a method that can globally monitor the oxidation of classical PTPs. We have now refined the q-oxPTPome approach, increasing its sensitivity by >10X. Applying q-oxPTPome to FH-deficient cell models shows that multiple PTPs are either highly oxidized (including PTPN12) or overexpressed. In general, highly oxidized PTPs were those that have relatively high sensitivity to exogenous H ₂ O ₂ . Most PTP oxidation in FH-deficient cells is reversible, although nearly 40% of PTPN13 is oxidized irreversibly to the sulfonic acid state. Using “substrate-trapping mutants”, we mapped PTPs to their putative substrates, and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identify PTPN12 as the major ABL1 phosphatase in HLRCC. Overall, our results show that ROS-induced PTPN12 oxidation accounts for ABL1 phosphorylation in HLRCC-associated PRCC, reveal a novel mechanism for inactivating a tumor suppressor gene product, and establish a direct link between pathological PTP oxidation and neoplastic disease.