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Purpose: Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases. Experimental Design: scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival. Results: The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression. Conclusions: Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Immunotherapy and targeted therapy are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of immunotherapy (IT; anti-PD1)->targeted therapy (TT; ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS- (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT->TT. Tumor volumes were measured and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-Seq), and reverse phase protein analysis (RPPA). scRNA-Seq demonstrated the IT->TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells (DCs) and natural killer (NK) cells, and decreased numbers of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDCSs), and regulatory T cells (Tregs). Durable responses to the IT->TT sequence were dependent on T-cell activity, with depletion of CD8(+), but not CD4(+) T cells, abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT->TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT->TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront immunotherapy improves the responses to targeted therapy in BRAF- and NRAS-mutant melanoma models.

Melanoma brain metastases (MBM) and leptomeningeal metastases (LMM) are two manifestations of melanoma dissemination to the CNS with vastly different survival outcomes. Analysis of single cell RNA-Seq data from 43 clinical specimens has uncovered a distinct, immune-suppressed T cell landscape in the LMM microenvironment that is distinct to those of the brain and skin metastases. An LMM patient with an extraordinarily long survival and documented response to therapy demonstrated an immune repertoire that was distinct from those of typical poor survivors and more similar to CSF from non-LMM donors. Analysis of serial specimens over the course of therapy demonstrated reductions in melanoma cells and macrophages, coupled with increased levels of T cells and dendritic cells in the CSF of the extraordinary responder, whereas poor survivors showed no improvement in T cell responses. In MBM patients, targeted therapy and immunotherapy was associated with increased immune infiltrate, with similar T cell transcriptional diversity noted between skin metastases and MBM - suggestive of immune cell trafficking into the brain. Treatment with targeted therapy was associated with an enrichment of CD8 T cells. Immunotherapy was associated with a more diverse lymphocyte landscape and higher numbers of antibody-producing cells. These findings were confirmed by multiplexed staining of patient specimens and using an immune-competent mouse model of MBM. Correlation analysis across the entire immune landscape identified the presence of a rare, novel population of dendritic cells (DC3s) to be correlated with increased overall survival, regardless of disease site/treatment. The presence of DC3s positively regulated the immune environment of both patient samples and preclinical melanoma models through modulation of activated T cells and MHC expression in the tumor. Our study provides the first comprehensive atlas of two distinct sites of melanoma CNS metastases and identifies rare populations of cells that underlie the biology of this devastating disease.

e16558 Background: Cyclooxygenase (COX) inhibition is postulated to restore the immune environment and synergizes with immune checkpoint inhibitors (ICI). The concurrent use of COX inhibitors (COXi) and ICI was associated with longer disease control in metastatic melanoma and non-small cell lung cancer. However, its role in mRCC remains unclear. Methods: We retrospectively reviewed 194 patients with mRCC treated with ICI (PD-(L)1 inhibitors +/- CTLA-4 inhibitors or TKIs) at Moffitt Cancer Center between 6/2014-7/2019. Concurrent use of COXi (aspirin [ASA] or NSAIDs) was defined as at least 3 weeks of COXi use during the first ICI course. Clinical characteristics of both arms were compared using Chi-squared or Kruskal-Wallis Rank Sum test. Time to progression (TTP) and Overall survival (OS) were compared using Kaplan Meier’s estimates. Univariate and multivariate Cox proportional hazards model was performed to evaluate the association between clinical factors, TTP, and OS. Results: Of 194 patients, 126 patients (64.9%) took COXi. Median age was 59.7 years and 80.4% were male. COXi arm had fewer patients with < 1 year from diagnosis to systemic treatment (45.9% vs 69.5%, p = 0.006) and more advanced age (median: 66 years vs 60 years, p = 0.01). IDMC risk group, number of prior therapies, neutrophil to lymphocytes ratio were similar between both arms. Median TTP was 8 months (m) for COXi arm and 12m for ICI only (HR 1.38; 95% CI [0.98, 1.94]). Median OS was 27m for COXi arm and 33m for ICI only (HR 1.05, 95% CI [0.69, 1.59]). Early mortality rate (within 3m of ICI treatments) were similar between both arms. Conclusions: In contrast to melanoma and lung cancer, concurrent use of COXi and ICI did not improve TTP and OS in patients with mRCC. The dual blockade showed a trend for shorter TTP. Further validation studies with larger cohorts are needed to confirm this finding.[Table: see text]

Purpose: Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Experimental Design: Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. To describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry–based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results: Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, whereas DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions: Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology among kidney cancer subtypes. Clin Cancer Res; 22(22); 5605–16. ©2016 AACR.

Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.

The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlating with activating mutations while three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172 and pY1197) were inhibited by erlotinib in concentration-dependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation.

The epidermal growth factor receptor (EGFR) is a major therapeutic target in metastatic colorectal cancer (CRC). Mutations in KRAS/NRAS have been identified as the resistance mechanisms to anti-EGFR antibody therapy (cetuximab/pantiumab). However, genomic understanding of the sensitivity to the EGFR-targeted therapy is lacking. A pre-specified, 203-gene cetuximab sensitivity (CTX-S) expression signature score was first validated on two previously published datasets: (1) 77 CTX-treated CRC tumors (progression free survival (PFS)>7 days), and (2) 147 in vitro CTX-treated CRC cell lines. The CTX-S signature was found to be consistently correlated with a newly developed 24-gene APC (truncating) mutations-specific Wnt/β-catenin score on four independent CRC datasets that also include Moffitt (n=468) and TCGA (n=624) CRC datasets. This promoted us to carry out a variety of analyses that revealed a cooperative role of mutant APC + mutant TP53 in identifying cetuximab sensitive subpopulations, which was further evidenced by additional validation analysis on two CRC PDX datasets (n=52 and n=98). While debating or inconclusive, other potential predictive markers of EGFRi therapies have been reported, including BRAF(V600E), PIK3CA mutations, loss of PTEN, and overexpression of HER2 as negative indicators, as well as increased EGFR copy number, and overexpression of EGFR ligands epiregulin and amphiregulin as positive indicators. Here, for the first time, we report that the mutant genes may cooperatively act as positive indicators of EGFRi response. Our study suggests that addition of the routine sequencing of APC and TP53 to extended RAS testing may refine the patient selection strategy to improve cetuximab/pantiumab response and potentially expand the therapeutic opportunity to previously-excluded mutant KRAS/NRAS patients who have “APC + TP53” mutations. Citation Format: Mingli Yang, Michael J. Schell, Andrey Loboda, Michael V. Nebozhyn, Jiannong Li, Jamie K. Teer, W. Jack Pledger, Timothy J. Yeatman. An integral genomic/expression analysis reveals a cooperative role of APC and TP53 mutations in identifying cetuximab-sensitive colorectal cancer [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 2571.

We describe a strategy for comprehending signaling pathways that are active in lung cancer cells and that are targeted by dasatinib using chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine-phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SRC-family kinase (SFK) members (LYN, SRC, FYN, LCK and YES), nonreceptor tyrosine kinases (FRK, BRK and ACK) and receptor tyrosine kinases (Ephrin receptors, DDR1 and EGFR). Using quantitative phosphoproteomics, we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug-resistant gatekeeper mutants, we show that SFKs (particularly SRC and FYN), as well as EGFR, are relevant targets for dasatinib action. The combined mass spectrometry-based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and a rationale for combinatorial therapeutic strategies.

Activating mutations in the epidermal growth factor receptor (EGFR) selectively activate signal transducers and activators of transcription (STAT) and Akt survival signaling pathways important in lung cancer cell growth and survival. Many kinases, such as EGFR, rely on heat shock protein 90 (Hsp90) chaperone function for conformational maturation and proper function. Histone deacetylase inhibitors (HDACi) have been suggested to regulate signaling protein interactions via modulation of protein chaperone function through Hsp90. For these reasons, we evaluated the effect of a HDACi in lung cancer cells with defined EGFR status. Cell lines with defined EGFR status and sensitivity to EGFR tyrosine kinase inhibitors were exposed to the HDACi LBH589, and the effects on cell survival, proliferation, and downstream signaling were evaluated. LBH589 resulted in increased acetylation of Hsp90 and reduced association of Hsp90 with EGFR, Akt, and STAT3. LBH589 selectively depleted proteins important in signaling cascades in cell lines harboring EGFR kinase mutations, such as EGFR, STAT3, and Akt, and these cells underwent apoptosis following exposure to LBH589. In addition, we found depletion of STAT3-dependent survival proteins, including Bcl-xL, Mcl-1, and Bcl-2. Conversely, LBH589 had little effect on apoptosis in cells not dependent on EGFR for survival, no changes were identified in the expression of EGFR or other survival proteins, and the predominant effect was cell cycle arrest rather than apoptosis. A 10-fold increase in LBH589 was necessary to observe durable depletion of EGFR and Akt in cells not harboring EGFR mutation. Treatment of cells with erlotinib and LBH589 resulted in synergistic effects on lung cancer cells dependent on EGFR for growth and/or survival. Based on these results, LBH589 can acetylate Hsp90, deplete EGFR and other key survival signaling proteins, and trigger apoptosis only in lung cancer cells harboring EGFR mutations. Therefore, EGFR mutation status may be predictive of outcome with LBH589 and possibly other HDACi. [Mol Cancer Ther 2007;6(9):2515–24]

Overexpression of Bcl-2 family members as well as deregulated apoptosis pathways are known hallmarks of lung cancer. Non-small cell lung cancer (NSCLC) cells are typically resistant to cytotoxic chemotherapy and approaches that alter the balance between pro-survival and pro-death Bcl-2 family members have shown promise in preclinical models of NSCLC. Here we evaluated the effects of a novel pan-Bcl-2 inhibitor GX15-070 on NSCLC survival and when combined with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors as well as traditional cytotoxic agents. GX15-070 is a small molecule agent that binds anti-apoptotic Bcl-2 proteins and interferes with their ability to interact with pro-apoptotic proteins. We evaluated the effect of GX15-070 and correlated the effect on EGFR status as well as Bcl-2 family protein expression. We show that GX15-070 can disrupt Mcl-1:Bak interactions in lung cancer cells. We identified differential sensitivity of a panel of lung cancer cells to GX15-070 and no clear relationship existed between EGFR status or Bcl-2 family protein expression and sensitivity to GX15-070. GX15-070 could induce apoptosis in a subset of lung cancer cell lines and this correlated with the effects on cell viability. GX15-070 combined with gefitinib was synergistic in a cell line dependent on EGFR for survival but GX15-070 could not reverse resistance to gefitinib in cell lines not dependent on EGFR for survival. Finally, we observed synergy between GX15-070 and cisplatin in NSCLC cells. Based on these results, GX15-070 can trigger apoptosis in NSCLC cells and can enhance chemotherapy-induced death. These data suggest that clinical trials with GX15-070 in combination with cytotoxic chemotherapy are indicated.

Stat transcription factors are activated by cytokines and can activate pathways important in oncogenesis. Although previous studies have identified an oncogenic role of Stat3 in lung cancer cells, the role of Stat1 is unclear. Using a mutant of Stat1 with constitutive activity (Stat1C), we examined the effect of persistent Stat1 activity on lung cancer cell growth, survival and gene expression. We identified no significant effect of Stat1C alone or with interferon-gamma (IFN-gamma) on lung cancer cell growth or survival. Consistent with prior reports, Stat1C expression alone elicited minimal changes in gene expression and required costimulatory IFN-gamma for full activity. Using oligonucleotide gene arrays and quantitative real-time PCR, we identified numerous proinflammatory gene products and chemokines regulated by IFN-gamma/Stat1C signaling. These results suggest the major role of IFN-gamma and Stat1 in lung cells is to direct a proinflammatory gene expression program rather than have major effects on cell growth or survival or both.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined. Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

In this study we show that via stable, retroviral-expression of tagged EGFR del (L747-S752 deletion mutant) in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a Flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR del complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material.

Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival. Cancer Res; 72(10); 2501–11. ©2012 AACR.

3-Bromopropionylamino benzoylurea (JIMB01) is a small molecular weight compound (MW 313) that has been synthesized in our laboratory. This compound showed antiproliferative activities in a panel of thirteen human tumor cell lines with ic 50 values in the range of 0.25 to 0.51 μM for leukemia and lymphoma cell lines and 0.33 to 9.26 μM for solid tumor cell lines. The primary action of JIMB01 is to inhibit microtubule polymerization but not depolymerization. A 4 μM concentration of the compound caused a complete inhibition of microtubule assembly in a cell-free reaction. An increase in the number of human hepatocarcinoma cells blocked in the M-phase was detected 12 hr after exposure to JIMB01. The kinase activity of cyclin B1, which is responsible for the G 2 /M transition, was increased accordingly. Bcl-2 phosphorylation became visible, in a western blot, within 6 hr in hepatocarcinoma cells treated with JIMB01 at 0.8 μM or higher. JIMB01-induced apoptosis in liver cancer cells was confirmed by morphological methods, flow cytometry, as well as DNA gel electrophoresis, which clearly demonstrated DNA degradation in the form of a multiple-unit DNA ladder. Furthermore, in vivo experiments using nude mice showed that intraperitoneal injection of JIMB01 at 15 mg/kg (with seven injections at 4-day intervals) significantly inhibited the growth of a human hepatocarcinoma (BEL-7402) by 66% in tumor volume ( P =0.01), at least compatible to the inhibition by vincristine (43% inhibition), indicating good bioavailability of the compound in the circulation. Side-effects of the compound were not observed, and the body weight of the treated mice remained stable during the 4-week treatment. Since JIMB01 is a small compound, targets a specific molecule in tumor cells, and has promising activity against human hepatocarcinoma in vivo , we believe JIMB01 merits consideration for further investigation.

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

KRAS mutations account for development of approximately 30% of lung adenocarcinomas. While RAF-MEK-ERK signaling cascade is a key regulator of RAS-mediated proliferation and survival, the clinical efficacy of MEK inhibitors is still underwhelming in RAS-driven cancers. We treated KRAS mutant non-small cell lung cancer (NSCLC) cell lines with two MEK inhibitors (AZD6244 and MEK162) currently in clinical development. In all cell lines tested (N=11), we failed to observe significant effects on cell viability or induction of apoptosis. Because increasing evidence indicates that cancer cells undergo adaptive proteomic rewiring in response to kinase inhibitors, we performed activity-based protein profiling (ABPP) after treating 11 KRAS mutant NSCLC cell lines with MEK inhibitors in order to identify global enzymatic changes potentially involved in these adaptive processes. Our ABPP approach employs desthiobiotin-ATP probe, which enables selective labeling of active sites in ATP-binding proteins including kinases, chaperones and metabolic enzymes. Labeled proteins were subsequently purified and analyzed by mass spectrometry-based proteomics technique. Our ABPP results revealed that MEK inhibitors increased levels of multiple receptor tyrosine kinases (RTK) including EGFR and MET. MEK inhibitors induced MET phosphorylation and EGF-mediated AKT phosphorylation was markedly enhanced in the presence of MEK inhibitors, indicating increased RTK activity in adaptive response to MEK inhibition. We also found that MEK inhibition led to increased multiple heat shock proteins including HSP90 and HSP70, suggesting the possibility of chaperone-mediated adaptive resistance. Finally, MEK inhibitors globally increased enzymes involved in glucose utilization pathways implicating metabolic alterations in adaptive response to MEK inhibition. Collectively, our systematic activity-based proteomic profiling approach provides novel insights into adaptive resistance mechanisms to MEK inhibitors, suggesting rational combination treatment strategies for KRAS mutant NSCLC. Updated works will be presented. Citation Format: Jae-Young Kim, Bin Fang, Eric A. Welsh, Fumi Kinose, Jiannong Li, Steven A. Eschrich, John M. Koomen, Eric B. Haura. H. Activity-based protein profiling reveals adaptive response to pharmacological MEK inhibition in KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A31. doi: 10.1158/1557-3125.RASONC14-A31

Introduction: The use of kinase inhibitors with targets such as VEGFR has heralded a new era of treatment for metastatic renal cell carcinoma (RCC) patients. However, patients develop resistance with time. Therefore, novel targets must be identified and a systems level description of RCC produced. To this end, we used mass spectrometry based phosphoproteomics to nominate targets as well as compare RCC subtypes. Methods: We developed an integrative strategy combining RCC phosphoproteomics and functional interrogation using drug screening. Tyrosine phosphorylated peptide immunoprecipitation and purification was performed in clear cell RCC (ccRCC) and normal renal cortex cell lines and analyzed via nano-liquid chromatography/tandem mass spectrometry interfaced with an electrospray hybrid ion-trap mass spectrometer. Eight ccRCC and five papillary RCC (pRCC) tumor tissues were also analyzed. We assembled a library of 180 small molecules, including 60 tyrosine kinase inhibitors (TKIs), and screened them at multiple concentrations in 10 clear cell RCC (ccRCC) cell lines. Results: Among the ccRCC cell lines, 153 phosphosites (114 proteins) were identified. Receptor tyrosine kinases included EGFR, MET, ERBB4, and EPHB2 and nonreceptor tyrosine kinases included ABL2, JAK2, KALRN, and PTK2. Serine/threonine kinases were the most abundant kinases and included, among others, several CDK and MAPK members. Other frequently observed phosphosites corresponded to cytoskeleton (10%) and cell adhesion (6%) proteins. Among the ccRCC tumors, 119 phosphosites (99 proteins) were identified. In contrast to the cell lines, the ccRCC tumors demonstrated increased Src family kinase signaling but less RTK signaling. Among the pRCC tumors, 113 phosphosites (98 proteins) were identified, including 37 not identified in the ccRCC tumors. This data, coupled with the TKI screen, offered some interesting observations. MET phosphotyrosine peptide intensity was relatively low in ccRCC compared to pRCC and among the eight MET inhibitors tested, seven failed to significantly inhibit cell viability. These results suggest that MET signaling may be more active in pRCC than ccRCC. Other divergent patterns of phosphotyrosine signaling emerged. For example, certain tyrosine phosphatases that could impact signaling displayed higher phosphorylation patterns in ccRCC. Other kinases currently not targeted by RCC therapeutics were more highly phosphorylated in pRCC relative to ccRCC tumors. The functional impact of these findings is currently undergoing evaluation. Conclusions: Integrating global phosphoproteomics of cell lines and tumors with functional analyses can shed light on key survival and growth pathways in clear cell and papillary RCC. Further studies in human tissues are ongoing and will be presented. Citation Format: Scott M. Haake, Jiannong Li, Yun Bai, Bin Fang, Julio Pow-Sang, John Koomen, Mayer Fishman, Eric B. Haura. Phosphoproteomics analysis reveals differential signaling pathways among clear cell and papillary renal cell carcinoma tumors. [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 5305. doi:10.1158/1538-7445.AM2014-5305

A ‘lung cancer'-specific mutant EGFR interactome was generated by a global analysis of protein–protein interactions and phosphorylation. After functional screening, nine proteins were identified as essential for the viability of EGFR-mutant lung cancer cells., The interactome of lung cancer-associated mutant forms of epidermal growth factor receptor (EGFR), consisting of 263 proteins, was built by integrating protein–protein interactions and tyrosine phosphorylation. Systematic perturbations of the network nodes revealed a core network of 14 proteins, 9 of which were shown to be specifically associated with survival of EGFR-mutant lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors showed differential dependence on the core network proteins. A drug network associated with the core network proteins led to the identification of two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib., We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.

Predictive biomarkers are critical to optimize the use of receptor tyrosine kinase (RTK) inhibitors for cancer therapy. Because multi-protein complexes are critical to maintain oncogenic signaling, we hypothesized that assays that measure protein-protein interactions could identify patients with RTK activity and predict RTK inhibitor response. We developed in situ proximity ligation assays (PLA) to assess the degree of active EGFR signaling in both non-small cell lung cancer (NSCLC) cell lines and formalin-fixed paraffin-embedded (FFPE) patient specimens with the goal of establishing PLA assays as biomarkers of erlotinib sensitivity. Using in situ PLA, we evaluated EGFR association with its signaling adaptors, GRB2 and SHC1. By confocal microscopy we observed increased PLA signals in EGFR mutant NSCLC cell lines (PC9, H1650, HCC4006, HCC827) relative to EGFR wildtype (H23, H1299, H322, A549), indicative of constitutively active signaling complexes in mutant cell lines. PLA signal intensity was independent of EGFR, GRB2 and SHC1 protein expression levels, but did correlate with co-IP and pEGFR immunoblot results. Erlotinib dose-dependently reduced PLA signals in PC9 cells. H1650 xenografts exhibited high PLA signals that were reduced by erlotinib, corresponding with tumor shrinkage. Conversely, low PLA signals were observed in xenografts from EGFR wildtype cell lines (H322, H157). In human xenografts, PLA signals were significantly higher in EGFR mutant xenografts and reduced by erlotinib. In human FFPE specimens, PLA signals were absent in normal non-epithelial tissues and low in epithelial portions. FFPE lung tumor specimens (N = 221) revealed a wide range of PLA signal intensities (scored as 0+ to 3+), which localized to cytokeratin-positive regions. High PLA signals were found in approximately 25% of specimens across multiple histological classifications, including adenocarcinoma, squamous and large cell carcinoma. Furthermore, specimens from metastatic brain lesions were highly enriched for high PLA signal (p2+) in specimens with relatively low EGFR protein expression and low PLA signal ( Citation Format: Matthew A. Smith, Richard Hall, Scott Haake, Jiannong Li, Eric B. Haura. Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity. [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 3502. doi:10.1158/1538-7445.AM2013-3502

Lung cancer is a devastating worldwide disease yet enthusiasm exists for treatment of subsets of the disease with molecularly targeted agents. Mutations in the epidermal growth factor receptor (EGFR) or translocation of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) define two unique subsets of lung cancer characterized by sensitivity to tyrosine kinase inhibitors (TKI). Despite striking results with TKI, not all patients respond, the drugs are non-curative, and resistance is universal. Mutations in KRAS also define a group of patients awaiting therapeutic opportunities. We are characterizing signaling networks using tandem affinity purification (TAP) and liquid chromatography-mass spectrometry (LC-MS/MS) to map protein-protein interactions (PPI) and anti-phosphotyrosine immunoprecipitation coupled with LC-MS/MS to map tyrosine phosphorylation. In PC9 cells with mutated EGFR, we characterized a physical EGFR network consisting of 266 proteins by integrating both TAP and pTyr MS data. In H3122 cells harboring EML4-ALK, we identified a PPI network consisting of 113 proteins and using pTyr MS identified changes in tyrosine phosphorylation in 120 proteins (58 decreased, 62 increased) following exposure to ALK tyrosine kinase inhibitor. Functional proteins are being discovered from these networks using siRNA and inhibitor screens. In parallel studies, we have exploited the use of chemical proteomics to discern targets of promiscuous kinase inhibitors and enable optimal combination approaches. In this way, we view network mapping linked to chemical proteomics as one approach to discern novel drug combination studies for in vivo validation and ultimately translation to early phase clinical trials. To translate PPI based mass spectrometry studies to clinic, we are developing in situ assays that identify and quantify PPI using proximal ligation assays (PLA). Pilot studies identify EGFR:Grb2 interactions in formalin fixed human lung cancers. These approaches have the potential to enable ‘network medicine’ by identifying novel combination approaches as well as through identifying subtypes of cancers through network views of cancer.

The translocation of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) defines a unique, new subset of patients with non small cell lung cancer (NSCLC). It represents a novel diagnostic biomarker and therapeutic target for NSCLC. PF 02341066 is a ALK small tyrosine kinase inhibitor that has demonstrated high clinical activity in EML4-ALK- positive NSCLC patients. We sought to characterize the signaling network used by EML4-ALK and reveal potential new therapeutic targets. We used tandem affinity purification (TAP) and liquid chromatography-mass spectrometry (LC-MS/MS) to map protein-protein interactions and anti-phosphotyrosine immunoprecipitation coupled with LC-MS/MS to map tyrosine phosphorylation. We selected the human NSCLC H3122 cell line for our studies as it is sensitive to PF 02341066 and harbors ELM4-ALK. Quantitative western blotting results show that activities of MET, ALK, AKT, ERK and STAT3 were inhibited by treatment with PF 02341066. Proteome-wide analysis of tyrosine phosphorylation of the ALK signaling pathway were examined. In total, 311 tyrosine phosphorylated proteins (487 unique phosphorylated sites) were identified. Of those, 73 pTyr sites (62proteins) were upregulated and 78 pTyr sites (58 proteins) were downregulated by 1uM PF02341066. Using TAP, we identified 13 proteints interacting with EML4-ALK including GRB2 and SHC1. Core interactions were verified by coupling of immunoprecipitation to Western blot and mass spectrometry. A comprehensive physical ALK network was generated by integrating both phosphoproteomic data and interactional partners using pathway analysis algorithms. Two adaptors, SHC1and GAB2, are suggested as the first outlets of signaling propagation from ALK. Other sublevels directly connected with ALK may allow further understanding the mechanism of ‘ALK addiction’. A sub-network composed of 35 cell death associated proteins was created. An ongoing functional analysis including siRNA and small inhibitor screen on this small network should validate druggable targets. This network approach has potential translational applications to identify novel approaches to treatment of ALK addicted lung cancers and could be useful in patients with acquired resistance to ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2011-1627

Goal: The goal of this experiment is to understand how kinase inhibitor therapies, like Erlotinib targeting the epidermal growth factor receptor (EGFR), modulate the signaling network using phosphoproteomics; developing a straightforward process to enable physicians to gather information that may assist in establishing a molecular basis for selection of chemotherapy, particularly small tyrosine inhibitor therapies, and for tracking patients’ responses to treatment. Methods: Non-small cell lung cancer (NSCLC) cell lines were used as model systems for examining lung cancer signaling, particularly the HCC827 cell line that overexpresses EGFR. To examine the response to inhibitor, EGFR from HCC827 cells was enriched by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation. After cell lysis, protein assays were carried out so that an equivalent amount of total protein was loaded into each lane. After excision of the appropriate region of the gel, reduction, and alkylation, in-gel trypsin digestion was performed. The resulting peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on a hybrid linear ion trap-orbital ion trap mass spectrometer. The high resolution and accurate mass measurements of the peptides enable relative quantification that can be used to examine dose response for each phosphorylation site. After detection and relative quantification of peptides including phosphorylation sites, quantitative assays based on liquid chromatography coupled to multiple reaction monitoring (LC-MRM) can be developed to increase sensitivity for subsequent translation to patient samples. Results: Using high resolution mass spectrometry, more than ten phosphotyrosine sites could be detected and quantified from EGFR and several of these phosphorylations were shown to be dose dependent to Erlotinib, a kinase inhibitor specific for EGFR. The decreases in phosphorylation at sites including tyrosine 1197 can be used as surrogates to monitor the inhibition of EGFR activity for determination of patient response. LC-MRM assays have been developed for EGFR expression and modification and implemented in the HCC827 cell line. Conclusion: The ability to monitor the effectiveness of the drug treatment at a molecular level allows physicians to modulate drug treatment based on the responses observed for each patient. This knowledge can maximize treatment effectiveness or suggest when other treatments may be more effective. The phosphoproteomics techniques illustrated here for the well characterized EGFR molecule can also be utilized to examine the drug induced molecular changes in other protein targets of clinical significance. Because of the potential importance of this experiment in clinical applications, a manuscript is being prepared for a chemical education journal that describes the lab activities and provides a virtual learning environment to monitor drug response with mass spectrometry. ∗This presenting student is supported by an ARRA supplement to the NCI Cancer Center Support Grant awarded to Moffitt (3P30 CA076292-11S6 PI WS Dalton) to create a training program for underrepresented undergraduate students in clinical proteomics Project LINK (Leaders In New Knowledge-Emerging Technologies) that also emphasizes education in health disparities and community outreach. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A68.