Reverse Phase Protein Array Analysis Research Articles

Abstract 1111: MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers

Abstract Targeted therapy of ROS1 fusion-driven non-small cell lung cancer (NSCLC) with the tyrosine kinase inhibitor (TKI), crizotinib, has achieved notable clinical success. Despite these promising results, resistance to therapy poses a significant challenge. 47-92% of crizotinib-resistant tumors have a wild-type ROS1 kinase domain suggesting that concurrent aberrations in bypass signaling pathways contribute to sustained cancer growth even in the presence of ROS1 inhibition. Bypass resistance pathways are not well-characterized in ROS1 fusion-driven cancers; hence there is a dearth of secondary therapeutic strategies. To identify recurrent genomic alterations that co-occur with ROS1 fusions and could potentially impinge on sensitivity to crizotinib and other emerging ROS1 TKIs, we interrogated MSK-IMPACT datasets and found that 10% of patients with ROS1-fusion have concurrent MYC copy number amplification (CNA). We hypothesized that c-Myc overexpression compensates for the loss of ROS1 signaling due to ROS1 TKI treatment. Here we dissected the role of c-Myc overexpression in diminishing ROS1 TKI sensitivity using two models: 1) a MYC-amplified and crizotinib-resistant patient-derived ROS1-fusion NSCLC cell line (LUAD0006) and 2) a CRISPR-Cas9 engineered human bronchial epithelial EZR-ROS1 cell line (HBEC-ER) exhibiting sensitivity to ROS1 TKI. We validated LUAD0006 ROS1 TKI-resistance using cell viability assays and confirmed MYC CNA and protein overexpression with fluorescence in situ hybridization and immunoblotting, respectively. Live cell imaging assays revealed that ROS1-TKI or c-Myc knockdown via shRNA alone were insufficient to inhibit LUAD0006 cell growth; however combined ablation of ROS1 kinase activity and c-Myc expression significantly suppressed cell proliferation. Gain of function experiments using HBEC-ER revealed that these cells acquire functional resistance to multiple ROS1 TKIs upon expression of a degradation-resistant c-Myc T58A mutant. Notably, immunoblotting confirmed 50-fold overexpression of c-Myc T58A relative to endogenous c-Myc in unmodified cells. We also performed RNA sequencing and reverse-phase protein array (RPPA) analysis of LUAD0006 cells treated with or without crizotinib and found that targets related to cell cycle, apoptosis, and metabolism were differentially expressed. Finally, we found that CDK4/6 and BET bromodomain inhibitors re-sensitized LUAD0006 cells to crizotinib, demonstrating the importance of c-Myc to the survival of these TKI-resistant cells. Taken together, we demonstrate that CNA or post-translational stabilization that leads to c-Myc overexpression confers resistance to crizotinib and other emerging ROS1 TKIs. These patients will likely benefit from a combination therapeutic strategy that targets both ROS1 and c-Myc. Citation Format: Sudarshan R. Iyer, Igor Odintsov, Adam J. Schoenfeld, Hiroki Sato, Evan Siau, Alexander Drilon, Gregory J. Riely, Romel Somwar, Marc Ladanyi, Monika A. Davare. MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1111.

Abstract 2867: Laboratory analyses of metastatic ER+/Her2- breast cancer treated with fulvestrant plus enzalutamide

Abstract Background: The clinical implications of the androgen receptor (AR), particularly in the context of aromatase inhibitor (AI) refractory metastatic breast cancer (MBC) are unclear. While AR is associated with more indolent primary tumors, high AR relative to ER in primary breast cancer is associated with endocrine resistance, and in the absence of estradiol or low or blocked ER, AR can exert a pro-survival signal. In a phase II trial of fulvestrant plus enzalutamide in ER+/Her2- MBC we analyzed serial biopsies pre- and post-treatment Methods: Eligible patients were women with ECOG 0-2, ER+/Her2- MBC. Fulvestrant 500 mg IM days 1, 15, 29 and every 4 weeks thereafter and Enzalutamide at 160 mg po daily on a continual basis were administered. Biopsies were required at study entry and at ~4 weeks on therapy. The clinical benefit rate at 24 weeks (CBR24) was the primary endpoint for efficacy. We performed mutational analysis to detect mutations including ESR1 exon 8 mutations.We examined estrogen, progesterone, androgen and glucocorticoid receptors, multiplex analysis of immune cells and PD-L1, and performed reverse phase protein array (RPPA) based protein pathway activation analysis of over 150 proteins/phosphoprotein drug targets from LCM-enriched tumor epithelium in baseline and post-treatment metastatic biopsies. Comparisons of PFS Responders (PFS longer than or equal to 24 weeks) and PFS Non-Responders (PFS shorter than or equal to 5 weeks) were performed using moderated t-tests on log2 transformed data. Results: 32 were eligible and median age was 61 years (46-87); PS 1 (0-1); a median of 2 prior chemotherapy and 2 prior hormonal therapies for metastatic disease (including 7 with prior Fulvestrant) and 90% had visceral disease. ESR1 mutant metastases had higher levels of ER and PR than those with wild type ESR1 (p<0.05). In a paired t test, ER and Ki67 decreased (p<0.05) with treatment. RPPA analysis of the baseline biopsy sample indicated activation of mTOR pathway proteins associated with non-response, while phosphorylated RB, EGFR and IRS1 were associated with response (PFS greater than 24 weeks). ESR1 mutation positive metastases had significantly more T helper cells, T regulatory cells and macrophages than those with wild type ESR1.PD-L1 increased with treatment in all patients by paired t test (p<0.03). Conclusions: Clinical benefit lasting 6-12 months was observed in 23% of patients. Our studies show important differences in hormone receptor expression and immune infiltrates in ESR1 mutated disease. RPPA based pathway activation mapping showed that mTOR pathway activation was associated with shorter PFS (p<0.05). Since PD-L1 expression significantly increased with treatment, we conclude that mTOR and/or PD-L1 directed therapies could be useful with Fulvestrant and Enzalutamide in patients with MBC refractory to current standard of care treatments Citation Format: Jennifer K. Richer, Nicole S. Spoelstra, Alyse Winchester, Julia Wulfkuhle, Sharon B. Sams, Gregory Vidal, Peter Kabos, Jennifer Diamond, Elena Shagisultanova, Anosheh Afghahi, Jose Mayordomo, Tessa McSpadden, Gloria Crawford, Virginia Borges, Dexiang Gao, Emanuel Petricoin, Anthony D. Elias. Laboratory analyses of metastatic ER+/Her2- breast cancer treated with fulvestrant plus enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2867.

Resistance Mechanisms Underlying Venetoclax Resistance in Mantle Cell Lymphoma

Background: BCL-2 is highly expressed in mantle cell lymphoma (MCL), and targeting BCL-2 with venetoclax (ABT-199) has resulted in positive clinical outcomes in MCL patients. However, resistance to various targeted agents often emerges; therefore, we generated venetoclax-resistant MCL cell lines to uncover the mechanisms mediating acquired venetoclax resistance and performed high-throughput drug screening to identify novel anti-MCL agents that can overcome venetoclax resistance.Methods: We generated 3 venetoclax-resistant cell lines (Mino, Rec-1 and Granta519) by long-term venetoclax exposure, and the anti-MCL activity of venetoclax against these acquired resistance lines was measured with dose-dependent cell viability and apoptosis assays compared with the parental lines. Aberrant protein expression between the parental and resistant cell lines was detected using Reverse Phase Protein Array (RPPA) analysis. A drug library (> 1000 drugs; Selleck Chemicals Ltd) was screened to identify agents that inhibit the cell growth of both the parental and resistant cells. The candidate drugs that showed anti-MCL activity were confirmed by dose-dependent cell viability and Annexin V apoptosis assays. To determine the mechanisms underlying the anti-MCL activity of the candidate agents in the parent and venetoclax-resistant MCL cell lines, we conducted RPPA analysis before and after drug treatment.Results: Chronic exposure to venetoclax resulted in markedly less sensitivity to venetoclax. The IC50 values of the venetoclax-resistant cell lines (Mino-Re, Rec-1-Re and Granta519-Re) were determined to be 603 nM, 676 nM and 456 nM, respectively, which are 600-fold, 500-fold and 25-fold higher than the IC50 values of the parental cells lines. RPPA analysis showed that proteins such as PTEN, PDCD4, AKT, ATM, BCL-2, BAX and BAK were down-regulated and p-AKT, p21, STAT3, Cyclin B1 and MCL-1 were up-regulated in the venetoclax-resistant MCL cell lines. The results were confirmed by Western blotting, and further Western blotting experiments revealed expression and modification differences among anti-apoptotic and pro-apoptotic protein family members between the parental and venetoclax-resistant MCL cell lines. Specifically, both MCL-1 and BCL-xL were elevated; however, BIM and BAX were absent in the venetoclax-resistant cells. High-throughput drug screening showed striking anti-MCL activity with two PI3K inhibitors, PIK-75 (PI3K α inhibitor) and GSK1059615 (pan PI3K inhibitor), in dose-dependent cell viability and apoptosis assays. RPPA analysis of the parental and venetoclax-resistant MCL cell lines with and without PI3K inhibitor treatment suggested that both drugs inhibited the cell viability of the MCL cell lines by blocking the activated PI3K/AKT pathway.Conclusion:The PI3K/AKT pathway is activated in venetoclax-resistant MCL cell lines, and targeting this pathway may be a potential approach to overcoming venetoclax resistance. DisclosuresWang:Pharmacyclics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Research Funding; BeiGene: Research Funding; Asana Biosciences: Research Funding; Proteolix: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma: Consultancy, Research Funding.

HnRNP K: A Driver of Myeloid Malignancy

Acute myeloid leukemia (AML) is increasingly defined as a heterogeneous group of malignancies divided largely by recurrent genetic and/or chromosomal aberrations. With this knowledge, development of targeted therapeutics has improved outcomes for some AML subsets. Increasing our understanding of alterations that drive disease development and progression will expand our armamentarium of therapeutic opportunities. To this end, our laboratory has identified a subset of AML that harbors increased expression of heterogeneous nuclear ribonucleoprotein K (HNRNPK; protein- hnRNP K).In a sizeable cohort of patients with AML, we have demonstrated HNRNPK gene amplification, RNA overexpression, and protein overexpression using FISH, qRT-PCR, and reverse phase protein array analyses. Importantly, the patient population with high protein expression of hnRNP K exhibited decreased median overall survival, increased WBC, and increased blast percentage in both bone marrow and peripheral blood. Together, these data suggest that hnRNP K may function as an oncogene in AML when overexpressed.To formally test this hypothesis, we generated a Vav-Cre;HnrnpkTg mouse model. Indeed, these mice overexpress hnRNP K in comparison to Vav-Cre mice or unrecombined littermates. Critically, Vav-Cre;HnrnpkTg mice develop myeloid malignancies. When transplanted, these malignancies have a decreased latency period and are rendered more aggressive. Currently, we are evaluating HSPC proliferation, differentiation potential, and using time of flight mass cytometry (CyTOF) to extensively characterize these transplanted leukemias.Functionally, hnRNP K is an RNA and ssDNA binding protein that can shuttle bidirectionally between the nucleus and cytoplasm. To further understand how hnRNP K can cause AML, we are evaluating subcellular localization of hnRNP K in the context of overexpression. As our preliminary experiments suggest a predominant cytoplasmic localization when hnRNP K is overexpressed, we are performing hnRNP K RNA-immunoprecipitation (RIP) followed by next generation sequencing in human cell lines with inducible overexpression of hnRNP K. In addition to confirming our previous observations of hnRNP K binding to the MYC transcript, we have identified several other putative targets for hnRNP K binding that may potentiate these malignant phenotypes.Additionally, we have performed RNA-Seq on HNRNPK- overexpressing human cell lines. These data suggest a significant role for hnRNP K in the regulation of innate immune response-observations that are consistent with phenotypes seen in our transplant models. Together, these studies provide insight into the pathways most critically altered in hnRNP K-overexpressing AML and will allow for appropriate utilization of therapeutic modalities. DisclosuresNo relevant conflicts of interest to declare.

Proteomic Profiling Identifies c-KIT and 53BP1 As Resistance Factors of AML Relapse Post Allogeneic Stem Cell Transplantation with G-CSF/Plerixafor/Busulfan-Fludarabine Conditioning

The CXCR4 inhibitor plerixafor in combination with G-CSF disrupts stroma-leukemia interactions, mobilizing leukemia cells from their niche microenvironment. This combination in conjunction with chemotherapy has been investigated in clinical trials in AML. We recently reported a phase 1/2 study of CXCR4 inhibition combined with chemotherapy prior to allogeneic stem cell transplantation (allo-SCT) (G-CSF-plerixafor (G+P) plus busulfan-fludarabine (Bu+Flu), Konopleva, et al. BMT 2015) and confirmed that patients who had not achieved remission at the time of transplant (“non-CR”) had poorer outcome than patients who were in remission at the time of allo-SCT (“CR”). Similar results have been reported in other clinical studies. Risk factors, such as the presence of circulating blasts and unfavorable cytogenetics are associated with poor outcome, but the molecular responses in patients treated with CXCR4 inhibitor and conditioning chemotherapy prior to allo-SCT are unknown. We investigated the hypothesis that the CXCR4 inhibition-driven preparative regimen modulates distinct signaling networks, which vary depending on disease status.Utilizing reverse phase protein array (RPPA), we profiled 157 proteins in 36 cellular signaling and functional groups in samples collected from ten AML patients who were studied in the phase 1/2 trial (Table1). Five CR patients had less than 1% blasts in bone marrow (BM) and no blasts in peripheral blood (PB) before conditioning regimen (baseline, day -9). Five non-CR patients had BM blasts ranging from 10-72% and PB blasts from 0-45%. Here we denote samples from “CR” patients as “normal” and from “non-CR” patient as “AML”. All “AML” patients with blasts had persistent blasts after G+P plus conditioning, ranging from 4-64%. However, four of five “AML” and five “normal” patients achieved complete response to allo-SCT. Engraftment was observed in all patients. Disease progression following allo-SCT was observed in four of five “AML” but not in “normal” patients. Overall survival in “AML” was significantly shorter than in “normal” patients (non-CR = 260 ± 55 days, CR = 2028 ± 103 days, P < 0.001).RPPA analysis of samples prior to conditioning (day -9) revealed that 9 proteins in 9 signaling pathways were expressed differently in “normal” and “AML” samples. Among them, c-KIT, 53BP1, a P53-binding DNA repair protein, and CIAP (a SMAC and IAP family member) had significantly higher expression in “AML”.G+P treatment significantly modulated 7 proteins in 4 pathways in “AML” samples, and 22 proteins in 13 signaling and functional groups in “normal” samples (Fig 1A). As a result, 22 proteins in 14 signaling and functional groups were differentially expressed in “AML” and “normal” samples post G+P treatment (day -7) (Fig 1B). Among the top 10 distinct proteins, PI3Kp110A, P53BP1, SMAD1 and p-C-JUN S73 had higher expression, and P38MAPK, p-HER2 Y1248, P-CADHERIN, SYK, P21 and TAU had lower expression in “AML” samples.Treatment with G+P plus Bu+Flu conditioning modulated 10 proteins in 7 signaling pathways in “AML” samples, and altered 40 proteins in 13 signaling pathways in “normal” samples (Fig 1C). As a result, G+P plus Bu+Flu conditioning caused significant differences in expression of 16 proteins in 14 signaling and functional groups in “AML” vs. “normal” samples (day -3) (Fig 1D). Among the top 10 distinct proteins, PI3Kp110A, XRCC1, p-ERA S118, 53BP1, and BCL2 had higher expression, and MIG6, SYK, TAU, p-AKT T308 and MEK1 had lower expression in “AML” samples. Notably, c-KIT and 53BP1 in “AML” baseline samples were persistently expressed at high levels in samples from G+P and G+P plus Bu+Flu treated patients, suggesting that c-KIT and 53BP1 mediated signaling might play a role in therapeutic resistance and disease relapse in patients with active disease at the time of transplant.In summary, CXCR4 inhibition combined with Bu+Flu conditioning triggered distinct molecular responses, resulting in differential signaling between “normal” and “AML” cells prior to allo-SCT. The persistence of c-KIT and 53BP1 might play a role in resistance and contribute to relapse in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

AMPK-regulated miRNA-210-3p is activated during ischaemic neuronal injury and modulates PI3K-p70S6K signalling.

Progressive neuronal injury following ischaemic stroke is associated with glutamate-induced depolarization, energetic stress and activation of AMP-activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR-210-3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR-210-3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3-phosphoinositide-dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR-210-3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over-activation in primary neurons, we demonstrated that induction of miR-210-3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR-210-3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR-210-3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

Abstract PS11-25: Pilot trial of priming with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency (DDRD) followed by cisplatin (cis) and nab paclitaxel (nab pac) in chemotherapy-pretreated metastatic triple negative breast cancer (metTNBC) pts

Abstract BACKGROUND: A subset of TNBCs have homologous recombination deficiency with upregulation of compensatory DNA repair pathways. The combination of TAK-228 and TAK-117 (PIKTOR), investigational oral TORC1/2 and PI3Kα selective inhibitors, respectively, is hypothesized to increase genomic instability (GI) and to increase DDRD, leading to increased sensitivity to DNA damaging chemotherapy and to checkpoint inhibitors in metTNBC pts. METHODS: 10 metTNBC pts received 4 mg PO TAK-228 and 200 mg PO TAK-117 QDx3d QW until disease progression (PD), followed by cis 75 mg/m2 plus nab pac 220 mg/m2 for up to 6 cycles. Primary endpoints were objective response rate with cis/nab pac and safety. Blood samples and biopsies of metastatic lesions were collected prior to and at PD on PIKTOR. Blood CTC analyses included enumeration, cell morphology, phenotypic heterogeneity, and predicted genomic instability (pGI) derived from cell phenotypes. RESULTS: 10 pts received PIKTOR followed by cis/nab pac. Median age: 51 yrs; median number of prior chemotherapy regimens was 3 (range, 1-5); 7 pts had prior carboplatin; sites of metastases: lymph nodes (n=8); lung (n=6); chest wall (n=1); bone (n=1); brain (n=1). Median time on PIKTOR was 8 wks (range, 3-14). With cis/nab pac, 1 pt had PR, 2 had SD &amp;gt; 6 mos, 1 had SD and 6 had PD. 2 SD pts (sites LNs and bone) and 1 PD pt (sites LNs), all carboplatin-pretreated, whose pre-PIKTOR BCs were PDL1-negative (2 pts) or unknown (1 pt) have durable SD on pembrolizumab post-cis/nab pac for 1+ yrs. PIKTOR related AEs ≥30% included: fatigue (90%); nausea (80%); diarrhea (60%); vomiting (40%); stomatitis (40%); hyperglycemia (30%); rash (30%); cough (30%); chest pain (30%). Incidence and grade of cis/nab pac-related AEs were not greater than expected with this regimen. At PD on PIKTOR, higher CTC burden and pGI+ CTCs were observed in a subset of pts suggesting that some CTCs may have developed greater GI with PIKTOR treatment. Next generation sequencing (NGS) and reverse phase protein array (RPPA) analyses of biomarkers of DNA repair pathways pre-and post-PIKTOR tissues are underway and will be presented at the meeting. CONCLUSIONS: Priming pts’ metTNBC with PIKTOR did not lead to durable responses with cis/nab pac in most pts in this pretreated population. However, 3/10 pts who had carboplatin-pretreated disease in LNs +/- bone, have highly durable SD on pembrolizumab following PIKTOR therapy. Citation Format: Joyce O'Shaughnessy, Esther San Roman Rodriguez, Priscilla Ontiveros, Rick Wenstrup, Tiziano Pramparo, Jessica Lang, Victoria Zismann, Natalia Briones, William Hendricks, Virginia Espina, Claudius Mueller, Maren K Levin. Pilot trial of priming with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency (DDRD) followed by cisplatin (cis) and nab paclitaxel (nab pac) in chemotherapy-pretreated metastatic triple negative breast cancer (metTNBC) pts [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-25.

Abstract PS4-07: Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference

Abstract Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P &amp;lt; 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P &amp;lt; 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Constitutive activation of mitogen-activated protein kinase kinase (MEK1) in ileal enterocytes leads to dysplasia and a predisposition to cancer.

Activation of mitogen-activated protein kinases (MAPKs) is a key factor in the pathogenesis of cancer, although the specific role of mitogen-activated protein kinase kinase (MEK1) is not well understood. Villin promoter-driven Cre expression was used to excise a floxed stop cassette from a phosphomimetically constitutively activated MEK1 (caMEK1) expression construct in the intestine of C57BL/6 mice. Zygosity status of caMEK1 afforded assessment of the dose dependence of the effect. The expected mendelian distribution of genotypes and sex was observed in 443 progenies. Between 21 and 63 days of life, caMEK1 had no effect on body weight in male mice, but reduced body weight in female mice homozygous for caMEK1. At 10 wk of age, the ileum of caMEK1-expressing mice was characterized by the finding of dysplasia and profound changes in overall architecture. Paneth cells were nearly absent in caMEK1 homozygotes. Targeted proteomic profiling via reverse phase protein array analyses with confirmatory Western blotting revealed significant changes in protein and phosphoprotein expression, including upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. Long-term viability of caMEK1 homozygous mice was reduced with no survival beyond 1 yr. Invasive adenocarcinoma developed in three of ten older mice [15 wk (homozygous), 26 wk (homozygous), and 35 wk (heterozygous) of age]. Expression of caMEK1 in enterocytes leads to marked derangements in the intestinal epithelium, which is associated with a predisposition to the development of invasive cancer.NEW & NOTEWORTHY The ileum of mice with constitutive expression of activated MEK1 (via phosphomimetic changes) in enterocytes is markedly abnormal with architectural distortion and cytologic atypia, which evolves into an adenoma invasive carcinoma sequence. Phosphoproteomic analysis reveals upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. This novel model provides new insights into intestinal homeostasis and carcinogenesis.

Multi-omic molecular profiling reveals potentially targetable abnormalities shared across multiple histologies of brain metastasis.

The deadly complication of brain metastasis (BM) is largely confined to a relatively narrow cross-section of systemic malignancies, suggesting a fundamental role for biological mechanisms shared across commonly brain metastatic tumor types. To identify and characterize such mechanisms, we performed genomic, transcriptional, and proteomic profiling using whole-exome sequencing, mRNA-seq, and reverse-phase protein array analysis in a cohort of the lung, breast, and renal cell carcinomas consisting of BM and patient-matched primary or extracranial metastatic tissues. While no specific genomic alterations were associated with BM, correlations with impaired cellular immunity, upregulated oxidative phosphorylation (OXPHOS), and canonical oncogenic signaling pathways including phosphoinositide 3-kinase (PI3K) signaling, were apparent across multiple tumor histologies. Multiplexed immunofluorescence analysis confirmed significant T cell depletion in BM, indicative of a fundamentally altered immune microenvironment. Moreover, functional studies using in vitro and in vivo modeling demonstrated heightened oxidative metabolism in BM along with sensitivity to OXPHOS inhibition in murine BM models and brain metastatic derivatives relative to isogenic parentals. These findings demonstrate that pathophysiological rewiring of oncogenic signaling, cellular metabolism, and immune microenvironment broadly characterizes BM. Further clarification of this biology will likely reveal promising targets for therapeutic development against BM arising from a broad variety of systemic cancers.

Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer.

Rationale: Little is known about the roles of proteoglycans in esophageal cancer. This study aims to investigate the roles and mechanisms of serglycin (SRGN) proteoglycan in promoting metastasis of esophageal squamous cell carcinoma (ESCC).Methods: Reverse phase protein array analysis was used to identify activated signaling pathways in SRGN-overexpressing cells. Chemokine array was used to identify differentially secreted factors from SRGN-overexpressing cells. Binding between SRGN and potential interacting partners was evaluated using proximity ligation assay and co-immunoprecipitation. The glycosaminoglycan (GAG) chains of SRGN were characterized using fluorophore-assisted carbohydrate electrophoresis. Tissue microarray and serum samples were used to determine the correlation of SRGN expression with clinicopathological parameters and patient survival.Results: In vitro and in vivo experiments showed that SRGN promoted invasion and metastasis in ESCC via activating ERK pathway, stabilizing c-Myc and upregulating the secretion of matrix metalloproteinases. SRGN-knockdown suppressed tumorigenic hallmarks. These SRGN-elicited functions were carried out in an autocrine manner by inducing the secretion of midkine (MDK), which was further identified as a novel binding partner of SRGN for the formation of a SRGN/MDK/CD44 complex. In addition, SRGN interacted with MDK and matrix metalloproteinase 2 in ESCC via its GAG chains, which were mainly decorated with chondroitin sulfate comprising of ∆di-4S and ∆di-6S CS. Clinically, high expression of serum SRGN in serum of patients with ESCC was an independent prognostic marker for poor survival.Conclusions: This study provides the first evidence that elevated serum SRGN has prognostic significance in patients with ESCC, and sheds light on the molecular mechanism by which elevated circulating SRGN in cancer patients might promote cancer progression.

Amphiregulin retains ERα expression in acquired aromatase inhibitor resistant breast cancer cells.

Acquired resistance to aromatase inhibitors (AIs) is a significant clinical issue in endocrine therapy for estrogen receptor (ER) positive breast cancer which accounts for the majority of breast cancer. Despite estrogen production being suppressed, ERα signaling remains active and plays a key role in most AI-resistant breast tumors. Here, we found that amphiregulin (AREG), an ERα transcriptional target and EGF receptor (EGFR) ligand, is crucial for maintaining ERα expression and signaling in acquired AI-resistant breast cancer cells. AREG was deregulated and critical for cell viability in ER+ AI-resistant breast cancer cells, and ectopic expression of AREG in hormone responsive breast cancer cells promoted endocrine resistance. RNA-sequencing and reverse phase protein array analyses revealed that AREG maintains ERα expression and signaling by activation of PI3K/Akt/mTOR signaling and upregulation of forkhead box M1 (FOXM1) and serum- and glucocorticoid-inducible kinase 3 (SGK3) expression. Our study uncovers a previously unappreciated role of AREG in maintaining ERα expression and signaling, and establishes the AREG-ERα crosstalk as a driver of acquired AI resistance in breast cancer.

Integrative analysis of multi-platform reverse-phase protein array data for the pharmacodynamic assessment of response to targeted therapies

Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.

Differences in gynecologic tumor development in Amhr2-Cre mice with KRASG12D or KRASG12V mutations

How different KRAS variants impact tumor initiation and progression in vivo has not been thoroughly examined. We hypothesize that the ability of either KRASG12D or KRASG12V mutations to initiate tumor formation is context dependent. Amhr2-Cre mice express Cre recombinase in tissues that develop into the fallopian tubes, uterus, and ovaries. We used these mice to conditionally express either the KRASG12V/+or KRASG12D/+ mutation. Mice with the genotype Amhr2-Cre Pten(fl/fl) KrasG12D/+(G12D mice) had abnormal follicle structures and developed low-grade serous ovarian carcinomas with 100% penetrance within 18 weeks. In contrast, mice with the genotype Amhr2-Cre Pten(fl/fl) KrasG12V/+ (G12V mice) had normal follicle structures, and about 90% of them developed uterine tumors with diverse histological features resembling those of leiomyoma and leiomyosarcoma. Granulosa cell tumors also developed in G12V mice. Differences in cell-signaling pathways in the uterine tissues of G12D and G12V mice were identified using RNA sequencing and reverse-phase protein array analyses. We found that CTNNB1, IL1A, IL1B, TNF, TGFB1, APP, and IL6 had the higher activity in G12V mice than in G12D mice. These mouse models will be useful for studying the differences in signaling pathways driven by KrasG12V/+ or KrasG12D/+ mutations to aid development of targeted therapies for specific KRAS mutant variants. Our leiomyoma model driven by the KrasG12V/+ mutation will also be useful in deciphering the malignant progression from leiomyoma to leiomyosarcoma.

Proteogenomic Analysis of Salivary Adenoid Cystic Carcinomas Defines Molecular Subtypes and Identifies Therapeutic Targets.

Salivary gland adenoid cystic carcinoma (ACC) has heterogeneous clinical behavior. Currently, all patients are treated uniformly, and no standard-of-care systemic therapy exists for metastatic ACC. We conducted an integrated proteogenomic analyses of ACC tumors to identify dysregulated pathways and propose a classification with therapeutic implications. RNA/DNA sequencing of 54 flash-frozen salivary ACCs and reverse phase protein array (RPPA) in 38 specimens were performed, with validation by Western blotting and/or IHC. Three independent ACC cohorts were used for validation. Both unbiased RNA sequencing (RNA-seq) and RPPA analysis revealed two molecular subtypes: ACC-I (37%) and ACC-II (63%). ACC-I had strong upregulation of MYC, MYC target genes, and mRNA splicing, enrichment of NOTCH-activating mutations, and dramatically worse prognosis. ACC-II exhibited upregulation of TP63 and receptor tyrosine kinases (AXL, MET, and EGFR) and less aggressive clinical course. TP63 and MYC were sufficient to assign tumors to ACC subtypes, which was validated in one independent cohort by IHC and two additional independent cohorts by RNA-seq. Furthermore, IHC staining for MYC and P63 protein levels can be used to identify ACC subtypes, enabling rapid clinical deployment to guide therapeutic decisions. Our data suggest a model in which ACC-I is driven by MYC signaling through either NOTCH mutations or direct amplification, which in turn suppress P63 signaling observed in ACC-II, producing unique therapeutic vulnerabilities for each subtype. Cooccurrence of multiple actionable protein/pathways alterations in each subtype indicates unique therapeutic vulnerabilities and opportunities for optimal combination therapy for this understudied and heterogeneous disease.

Rigosertib in Combination with Azacitidine Impacts Metabolic and Differentiation Pathways in the MDS-L Cell Line

Background Myelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis with peripheral blood cytopenias, leading to death from infection or bleeding. Azacitidine (AZA), a hypomethylating agent (HMA) is the standard of care for treatment of MDS patients (pts) with higher-risk MDS [Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017]. Responses to AZA occur in 50% of pts with significant effects on hematopoiesis ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence [Silverman LR, et al. Leukemia, 1993]. AZA treatment is associated with global DNA hypomethylation, including human endogenous retroviruses (HEV) which further activates innate immune signaling [Chiappinelli KB,et al. Cell, 2015]. The exact mechanism by which AZA improves hematopoiesis is unknown. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment with worsening cytopenias or transformation to leukemia [Silverman LR, et al. B. J Clin Oncol, 2002; Cancer, 2011]. Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical. Our clinical data demonstrate that AZA combined with Rigosertib (RIGO), a novel Ras mimetic that inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al. Cell, 2016], yields a response rate of 54% of pts who were HMA failures [Navada SC, et al. EHA 2019]. The response was associated with significant improvement in hematopoiesis and represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The precise mechanism that leads to reversal of the resistance phenotype is poorly understood. Methods: We investigated the differential protein expression in response to different treatment (AZA, RIGO alone and sequential combinations RIGO/AZA; AZA/RIGO) in vitro in MDS-L cell line by Reverse phase protein array (RPPA). Further, we also studied the functional role of these treatments on differentiation in the cell line by growing cells on semi-solid media as well as by flow cytometry using various stem cell and differentiation markers (CD34, CD38, CD45, CD123). Results: RPPA analysis indicated a discrete responses to treatment in the MDS-L cell line. The response was prominent with differential expression of 43 proteins specifically in MDS-L cells treated with RIGO/AZA that include PIK3R1, AKT1, mTOR, p38 MAPK, PTEN, RPS6KA1 (Fold change (FC) &amp;lt; -2). All of these proteins are downregulated which suggests inhibition of PI3K and mTOR signaling. Proteins belonging to metabolic pathways, including ACC1 and ACLY, were found to be downregulated, whereas proteins related to mitochondrial function and oxidative phosphorylation (OXPHOS) were upregulated (FC &amp;gt; 1.5/&amp;lt; -1.5) in cells treated with RIGO/AZA. In addition, we found that MDS-L cells represented both CD34+CD38+ and CD34+CD38- populations by FACS analysis (Fig 1). AZA increases the percentage of CD34+CD38+, indicative of differentiation, whereas RIGO alone increased the percentage of CD34+CD38- cells, representing a primitive stem cell population (Table in Fig 1). Based on the combination of differentiation markers (illustrated in fig 1), we observed that RIGO alone, and sequenced as combination RIGO/AZA, impacts different progenitors such as granulocyte-macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP), and multipotent progenitor (MPP). RIGO/AZA treatment showed a decrease in GMP, while the % of MEP was increased, as compared to other treatments. Moreover, we found a remarkable reduction in the number of colony forming units on differentiation media in response to RIGO (83%) and RIGO/AZA (90%). Conclusions: These data demonstrate that in addition to inhibition of the PI3/AKT/mTOR pathway, the RIGO/AZA combination also impacts metabolic and differentiation pathways of MDS-L cells. RIGO alone appears to promote maintenance of a primitive stem cell population, while the RIGO/AZA sequenced combination appears to push the cells toward a cycling stage with increased expression of genes associated with OXPHOS. In comparison, when treated with RIGO, cells remain in a less differentiated stage. Further studies are underway to determine the effect of metabolic changes on differentiation and maintenance of hematopoietic stem cells. Figure Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Research Funding. Silverman:Celgene: Research Funding; Medimmune: Research Funding; Onconova Therapeutics Inc: Patents &amp; Royalties, Research Funding.

Proteomics in Pediatric Acute Myeloid and T-Cell Lymphoblastic Leukemia: Shared Individual Protein Expression Patterns Co-Cluster into Overall Distinct Combinations

INTRODUCTION: Pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are heterogeneous diseases mediated by changes in protein expression. As most chemotherapeutic agents target proteins, and because overall survival of pediatric AML is far inferior to both pre-B and T-ALL, we aimed to compare the proteomic landscape of pediatric T-ALL and AML, with the goal of determining common AML-T-ALL pathways that are potentially targetable with novel agents. METHODS: Reverse phase protein arrays (RPPA) analysis was used to measure protein expression in 858 acute leukemia samples (358 T-ALL and 500 AML, 723 pediatric (&amp;lt; 18 yrs.), 135 adults (≥18 yrs.)) and 61 normal CD34+ samples using 270 validated antibodies. Expression levels were normalized against CD34+ cells. Proteins were allocated into 30 functionally related subgroups (Protein Functional Group (PFG)). A progeny clustering algorithm was applied to each PFG to search for strong correlations between proteins and to identify an optimal number of Protein Clusters (PC). Block clustering identified PC that recurrently co-occurred together (Protein Constellation (CON)) and patients that expressed similar combination of CON were defined as Protein Signature (SIG). Proteins that were differentially expressed were identified using the Student's t-test or ANOVA, with a Bonferroni adjusted p-value (0.05/ 270 = 0.00019)). RESULTS: Of the 270 analyzed proteins, 131 proteins (49%) were differentially expressed between T-ALL and AML; 60 were higher in T-ALL, 71 in AML. Similar to our previous analysis in adult AML and ALL, cell cycle regulators (CDKN1A, CDKN1B) and 2 of the 5 histone marks (H3K36Me3 &amp; H3K4Me3) were higher expressed in T-ALL compared to AML. Heat shock proteins (HSP90AA1_B1, HSPA1A_L, HSPB1 and HSPB1-pSer82) were higher in AML as well as translation proteins EIF2S1, EIF4E and EIF4EBP1 and ribosomal proteins RPS6-pSer235_236 and RPS6KB1, while expression of the translation inhibitory proteins EIF2S1-pSer51 and EIF2AK2-pThr451 was lower in AML compared to T-ALL. Next, cluster analysis in the context of 30 PFG resulted in 133 PC. The majority (n=102) of PC were expressed in both diseases, 30 PC (22.6%) were AML-specific, and only one PC was specific to T-ALL (characterized by high CDKN1A, CDKN1B and CCND1, but low WEE1, CCNB1 and RB1-pSer). Co-clustering of the 133 PC identified 14 CON that formed 17 SIG. Three CON (5, 9, 10) were associated with AML, 2 with T-ALL (2, 13) and 8 CON were observed in both diseases. In contrast, 15 of SIG were associated with either T-ALL or AML, and two SIG (9, 10) included a mixture of both diseases (P &amp;lt; 0.001, annotation bar Figure 1 "Disease")). SIG were associated with gender (P &amp;lt; 0.001), but not with CNS-status and ethnicity (Hispanic vs. non-Hispanic). No age-specific (kids vs. adults) signatures were observed. For each SIG and CON, proteins significantly higher or lower expressed compared to the normal CD34+ cells were identified. CONCLUSIONS: This study provides support for our previous hypothesis that pediatric T-ALL and AML can be characterized by recurrent protein expression patterns. While most PC and CON were found in both diseases, SIG (i.e. combinations of protein expression patterns) were specific to either T-ALL or AML. We found similar results when comparing B-ALL to AML in adults. Shared CON indicate that there are common protein expression patterns between pediatric T-ALL and AML. Proteins or pathways with similar utilization (e.g. CON3, 5) in both diseases may allow for information on clinical utility from one disease to be applicable to the other. Those with differential utilization are likely to be uninformative with respect to clinical utility in the other disease. Figure. "MetaGalaxy" analysis for pediatric AML and T-ALL. Each row represents one protein clusters (n = 133), each column represents one patient (n = 858). Blue indicates membership for that particular protein cluster. Annotation bar shows strong correlation with disease (yellow = T-ALL, blue = AML). No associations were seen for age (blue = adult, pink = pediatric) or Ethnicity (blue = Hispanic, yellow = non-Hispanic). Figure 1 Disclosures No relevant conflicts of interest to declare.

Phase 2 study of buparlisib (BKM120), a pan-class I PI3K inhibitor, in patients with metastatic triple-negative breast cancer

BackgroundTreatment options for triple-negative breast cancer remain limited. Activation of the PI3K pathway via loss of PTEN and/or INPP4B is common. Buparlisib is an orally bioavailable, pan-class I PI3K inhibitor. We evaluated the safety and efficacy of buparlisib in patients with metastatic triple-negative breast cancer.MethodsThis was a single-arm phase 2 study enrolling patients with triple-negative metastatic breast cancer. Patients were treated with buparlisib at a starting dose of 100 mg daily. The primary endpoint was clinical benefit, defined as confirmed complete response (CR), partial response (PR), or stable disease (SD) for ≥ 4 months, per RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. A subset of patients underwent pre- and on-treatment tumor tissue biopsies for correlative studies.ResultsFifty patients were enrolled. Median number of cycles was 2 (range 1–10). The clinical benefit rate was 12% (6 patients, all SD ≥ 4 months). Median PFS was 1.8 months (95% confidence interval [CI] 1.6–2.3). Median OS was 11.2 months (95% CI 6.2–25). The most frequent adverse events were fatigue (58% all grades, 8% grade 3), nausea (34% all grades, none grade 3), hyperglycemia (34% all grades, 4% grade 3), and anorexia (30% all grades, 2% grade 3). Eighteen percent of patients experienced depression (12% grade 1, 6% grade 2) and anxiety (10% grade 1, 8% grade 2). Alterations in PIK3CA/AKT1/PTEN were present in 6/27 patients with available targeted DNA sequencing (MSK-IMPACT), 3 of whom achieved SD as best overall response though none with clinical benefit ≥ 4 months. Of five patients with paired baseline and on-treatment biopsies, reverse phase protein arrays (RPPA) analysis demonstrated reduction of S6 phosphorylation in 2 of 3 patients who achieved SD, and in none of the patients with progressive disease.ConclusionsBuparlisib was associated with prolonged SD in a very small subset of patients with triple-negative breast cancer; however, no confirmed objective responses were observed. Downmodulation of key nodes in the PI3K pathway was observed in patients who achieved SD. PI3K pathway inhibition alone may be insufficient as a therapeutic strategy for triple-negative breast cancer.Trial registrationNCT01790932. Registered on 13 February 2013; NCT01629615. Registered on 27 June 2012.

Evaluation of the RAS signaling network in response to MEK inhibition using organoids derived from a familial adenomatous polyposis patient

RAS signaling is a promising target for colorectal cancer (CRC) therapy, and a variety of selective inhibitors have been developed. However, their use has often failed to demonstrate a significant benefit in CRC patients. Here, we used patient-derived organoids (PDOs) derived from a familial adenomatous polyposis (FAP) patient to analyze the response to chemotherapeutic agents targeting EGFR, BRAF and MEK. We found that PDOs carrying KRAS mutations were resistant to MEK inhibition, while those harboring the BRAF class 3 mutation were hypersensitive. We used a systematic approach to examine the phosphorylation of RAS effectors using reverse-phase protein array (RPPA) and found increased phosphorylation of MEK induced by binimetinib. A high basal level of ERK phosphorylation and its rebound activation after MEK inhibition were detected in KRAS-mutant PDOs. Notably, the phosphorylation of EGFR and AKT was more closely correlated with that of MEK than that of ERK. Transcriptome analysis identified MYC-mediated transcription and IFN signaling as significantly correlated gene sets in MEK inhibition. Our experiments demonstrated that RPPA analysis of PDOs, in combination with the genome and transcriptome, is a useful preclinical research platform to understand RAS signaling and provides clues for the development of chemotherapeutic strategies.