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PARP1 inhibitors (PARPis) are used for treatment of cancers with mutations in BRCA1 or BRCA2 that are deficient in homologous recombination. The identification of modulators of PARP1 activity is critical to understand and overcome resistance to PARPis. We integrated data from three omics-scale screens to discover new regulators of PARP1 activity. We identified SART1 and show that its silencing leads to an increase in poly-ADP ribosylation and chromatin-bound PARP1. SART1 is recruited to chromatin following DNA damage and limits PARP1 chromatin retention and activity. The SART1 N-terminus is sufficient to regulate the accumulation of PAR chains and PARP1 on chromatin, an activity dependent on the RGG/RG box. Silencing of SART1 leads to an increased sensitivity of cells to DNA damage induced by IR, irrespective of BRCA1 status and to PARPis only in absence of BRCA1 . These results suggest that SART1 could be clinically utilized to improve PARPi efficacy., Competing Interests: The authors state that they have no conflict of interest to disclose., (© 2024 The Author(s).)

Cancer-associated fibroblasts (CAFs) are associated with tumor progression and modulate drug sensitivity of cancer cells. However, the underlying mechanisms are often incompletely understood and crosstalk between tumor cells and CAFs involves soluble secreted as well as adhesion proteins. Interrogating a panel of non-small cell lung cancer (NSCLC) cell lines driven by EML4-ALK fusions, we observed substantial CAF-mediated drug resistance to clinical ALK tyrosine kinase inhibitors (TKIs). Array-based cytokine profiling of fibroblast-derived conditioned- media identified HGF-MET signaling as a major contributor to CAF-mediated paracrine resistance that can be overcome by MET TKIs. However, 'Cell Type specific labeling using Amino acid Precursors' (CTAP)-based expression and phosphoproteomics in direct coculture also highlighted a critical role for the fibronectin-integrin pathway. Flow cytometry analysis confirmed activation of integrin β1 (ITGB1) in lung cancer cells by CAF coculture. Treatment with pharmacological inhibitors, cancer cell-specific silencing or CRISPR-Cas9-mediated knockout of ITGB1 overcame adhesion protein-mediated resistance. Concurrent targeting of MET and integrin signaling effectively abrogated CAF-mediated resistance of EML4-ALK -driven NSCLC cells to ALK TKIs in vitro . Consistently, combination of the ALK TKI alectinib with the MET TKI capmatinib and/or the integrin inhibitor cilengitide was significantly more efficacious than single agent treatment in suppressing tumor growth using an in vivo EML4-ALK -dependent allograft mouse model of NSCLC. In summary, these findings emphasize the complexity of resistance-associated crosstalk between CAFs and cancer cells, which can involve multiple concurrent signaling pathways, and illustrate how comprehensive elucidation of paracrine and juxtacrine resistance mechanisms can inform on more effective therapeutic approaches.

Inactivating mutations in PTEN are prevalent in melanoma and are thought to support tumor development by hyperactivating the AKT/mTOR pathway. Conversely, activating mutations in AKT are relatively rare in melanoma, and therapies targeting AKT or mTOR have shown disappointing outcomes in preclinical models and clinical trials of melanoma. This has led to the speculation that PTEN suppresses melanoma by opposing AKT-independent pathways, potentially through noncanonical functions beyond its lipid phosphatase activity. In this study, we examined the mechanisms of PTEN-mediated suppression of melanoma formation through the restoration of various PTEN functions in PTEN-deficient cells or mouse models. PTEN lipid phosphatase activity predominantly inhibited melanoma cell proliferation, invasion, and tumor growth, with minimal contribution from its protein phosphatase and scaffold functions. A drug screen underscored the exquisite dependence of PTEN-deficient melanoma cells on the AKT/mTOR pathway. Furthermore, activation of AKT alone was sufficient to counteract several aspects of PTEN-mediated melanoma suppression, particularly invasion and the growth of allograft tumors. Phosphoproteomics analysis of the lipid phosphatase activity of PTEN validated its potent inhibition of AKT and many of its known targets, while also identifying the AP-1 transcription factor FRA1 as a downstream effector. The restoration of PTEN dampened FRA1 translation by inhibiting AKT/mTOR signaling, and FRA1 overexpression negated aspects of PTEN-mediated melanoma suppression akin to AKT. This study supports AKT as the key mediator of PTEN inactivation in melanoma and identifies an AKT/mTOR/FRA1 axis as a driver of melanomagenesis., Significance: PTEN suppresses melanoma predominantly through its lipid phosphatase function, which when lost, elevates FRA1 levels through AKT/mTOR signaling to promote several aspects of melanomagenesis., (©2024 American Association for Cancer Research.)

Purpose: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi., Experimental Design: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models., Results: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy., Conclusions: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination., (©2023 American Association for Cancer Research.)

BRCA1/2-deficient ovarian carcinoma (OC) has been shown to be particularly sensitive to poly (ADP-ribose) polymerase inhibitors (PARPis). Furthermore, BRCA1/2 mutation status is currently used as a predictive biomarker for PARPi therapy. Despite providing a major clinical benefit to the majority of patients, a significant proportion of BRCA1/2-deficient OC tumors do not respond to PARPis for reasons that are incompletely understood. Using an integrated chemical, phospho- and ADP-ribosylation proteomics approach, we sought here to develop additional mechanism-based biomarker candidates for PARPi therapy in OC and identify new targets for combination therapy to overcome primary resistance. Using chemical proteomics with PARPi baits in a BRCA1-isogenic OC cell line pair, as well as patient-derived BRCA1-proficient and BRCA1-deficient tumor samples, and subsequent validation by coimmunoprecipitation, we showed differential PARP1 and PARP2 protein complex composition in PARPi-sensitive, BRCA1-deficient UWB1.289 (UWB) cells compared to PARPi-insensitive, BRCA1-reconstituted UWB1.289+BRCA1 (UWB+B) cells. In addition, global phosphoproteomics and ADP-ribosylation proteomics furthermore revealed that the PARPi rucaparib induced the cell cycle pathway and nonhomologous end joining (NHEJ) pathway in UWB cells but downregulated ErbB signaling in UWB+B cells. In addition, we observed AKT PARylation and prosurvival AKT-mTOR signaling in UWB+B cells after PARPi treatment. Consistently, we found the synergy of PARPis with DNAPK or AKT inhibitors was more pronounced in UWB+B cells, highlighting these pathways as actionable vulnerabilities. In conclusion, we demonstrate the combination of chemical proteomics, phosphoproteomics, and ADP-ribosylation proteomics can identify differential PARP1/2 complexes and diverse, but actionable, drug compensatory signaling in OC., Competing Interests: Conflict of interest J. M. K. reports support from Bristol-Myers Squibb on an unrelated project. All other authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

PARP inhibitors (PARPis) display single-agent anticancer activity in small cell lung cancer (SCLC) and other neuroendocrine tumors independent of BRCA1/2 mutations. Here, we determine the differential efficacy of multiple clinical PARPis in SCLC cells. Compared with the other PARPis rucaparib, olaparib, and niraparib, talazoparib displays the highest potency across SCLC, including SLFN11-negative cells. Chemical proteomics identifies PARP16 as a unique talazoparib target in addition to PARP1. Silencing PARP16 significantly reduces cell survival, particularly in combination with PARP1 inhibition. Drug combination screening reveals talazoparib synergy with the WEE1/PLK1 inhibitor adavosertib. Global phosphoproteomics identifies disparate effects on cell-cycle and DNA damage signaling thereby illustrating underlying mechanisms of synergy, which is more pronounced for talazoparib than olaparib. Notably, silencing PARP16 further reduces cell survival in combination with olaparib and adavosertib. Together, these data suggest that PARP16 contributes to talazoparib's overall mechanism of action and constitutes an actionable target in SCLC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Theoretically, small molecule CDK4/6 inhibitors (CDK4/6is) represent a logical therapeutic option in non-small cell lung cancers since most of these malignancies have wildtype RB, the key target of CDKs and master regulator of the cell cycle. Unfortunately, CDK4/6is are found to have limited clinical activity as single agents in non-small cell lung cancer. To address this problem and to identify effective CDK4/6i combinations, we screened a library of targeted agents for efficacy in four non-small cell lung cancer lines treated with CDK4/6 inhibitors Palbociclib or Abemaciclib. The pan-PAK (p21-activated kinase) inhibitor PF03758309 emerged as a promising candidate with viability ratios indicating synergy in all 4 cell lines and for both CDK4/6is. It is noteworthy that the PAKs are downstream effectors of small GTPases Rac1 and Cdc42 and are overexpressed in a wide variety of cancers. Individually the compounds primarily induced cell cycle arrest; however, the synergistic combination induced apoptosis, accounting for the synergy. Surprisingly, while the pan-PAK inhibitor PF03758309 synergizes with CDK4/6is, no synergy occurs with group I PAK inhibitors FRAX486 or FRAX597. Cell lines treated only with Ribociclib, FRAX486 or FRAX597 underwent G1/G0 arrest, whereas combination treatment with these compounds predominantly resulted in autophagy. Combining high concentrations of FRAX486, which weakly inhibits PAK4, and Ribociclib, mimics the autophagy and apoptotic effect of PF03758309 combined with Ribociclib. FRAX597, a PAKi that does not inhibit PAK4 did not reduce autophagy in combination with Ribociclib. Our results suggest that a unique combination of PAKs plays a crucial role in the synergy of PAK inhibitors with CDK4/6i. Targeting this unique PAK combination, could greatly improve the efficacy of CDK4/6i and broaden the spectrum of cancer treatment., Competing Interests: The authors have declared that no competing interests exist.

Rearranged during transfection ( RET ) rearrangements occur in 1% to 2% of lung adenocarcinomas as well as other malignancies and are now established targets for tyrosine kinase inhibitors. We developed three novel RET fusion-positive ( RET +) patient-derived cancer cell lines, CUTO22 [kinesin 5B ( KIF5B )- RET fusion], CUTO32 ( KIF5B - RET fusion), and CUTO42 (echinoderm microtubule-associated protein-like 4- RET fusion), to study RET signaling and response to therapy. We confirmed each of our cell lines expresses the RET fusion protein and assessed their sensitivity to RET inhibitors. We found that the CUTO22 and CUTO42 cell lines were sensitive to multiple RET inhibitors, whereas the CUTO32 cell line was >10-fold more resistant to three RET inhibitors. We discovered that our RET + cell lines had differential regulation of the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B (AKT) pathways. After inhibition of RET, the CUTO42 cells had robust inhibition of phosphorylated AKT (pAKT), whereas CUTO22 and CUTO32 cells had sustained AKT activation. Next, we performed a drug screen, which revealed that the CUTO32 cells were sensitive (<1 nM IC 50 ) to inhibition of two cell cycle-regulating proteins, polo-like kinase 1 and Aurora kinase A. Finally, we show that two of these cell lines, CUTO32 and CUTO42, successfully establish xenografted tumors in nude mice. We demonstrated that the RET inhibitor BLU-667 was effective at inhibiting tumor growth in CUTO42 tumors but had a much less profound effect in CUTO32 tumors, consistent with our in vitro experiments. These data highlight the utility of new RET+ models to elucidate differences in response to tyrosine kinase inhibitors and downstream signaling regulation. Our RET+ cell lines effectively recapitulate the interpatient heterogeneity observed in response to RET inhibitors and reveal opportunities for alternative or combination therapies. SIGNIFICANCE STATEMENT: We have derived and characterized three novel rearranged during transfection (RET) fusion non-small cell lung cancer cell lines and demonstrated that they have differential responses to RET inhibition as well as regulation of downstream signaling, an area that has previously been limited by a lack of diverse cell line modes with endogenous RET fusions. These data offer important insight into regulation of response to RET tyrosine kinase inhibitors and other potential therapeutic targets., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: Covalent inhibitors of KRAS G12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRAS G12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear., Experimental Design: We performed mass spectrometry-based phosphoproteomics analysis in KRAS G12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type-specific markers, we compared proteome and phosphoproteomes of KRAS G12C cells. Gene expression patterns of KRAS G12C cell lines and lung tumor tissues were examined., Results: Our analysis suggests cell type-specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRAS G12C , we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures., Conclusions: Our phosphoproteomic study identified cell type-adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi., (©2021 American Association for Cancer Research.)

Activating mutations in BRAF, a key mediator of RAS signaling, are present in approximately 50% of melanoma patients. Pharmacologic inhibition of BRAF or the downstream MAP kinase MEK is highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Here, we show that treatment of melanoma patients with BRAF and MEK inhibitors (MEKi) activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. In both melanoma and lung cancer cells, MEKi increased cell-surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Screening of 289 targeted agents for the ability to increase TNFα and IFNγ target gene expression demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell-cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the antitumor efficacy of MEKi, whereas the administration of TNFα and IFNγ in MEKi-treated mice enhanced the antitumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. These findings define a novel cytokine response modulatory function of MEKi that can be therapeutically exploited. SIGNIFICANCE: Lung cancer cells are rendered sensitive to MEK inhibitors by TNFα and IFNγ, providing a strong mechanistic rationale for combining immunotherapeutics, such as checkpoint blockers, with MEK inhibitor therapy for lung cancer. See related commentary by Havel, p. 5699 ., (©2019 American Association for Cancer Research.)

Lung cancer patients with mutations in epidermal growth factor receptor (EGFR) benefit from treatments targeting tyrosine kinase inhibitors (TKIs). However, both intrinsic and acquired resistance of tumors to TKIs are common, and EGFR variants have been identified that are resistant to multiple TKIs. In the present study, we characterized selected EGFR variants previously observed in lung cancer patients and expressed in a murine bone marrow pro-B Ba/F3 cell model. Among these EGFR variants, we report that an exon 20 deletion/insertion mutation S768insVGH is resistant to erlotinib (a first-generation TKI), but sensitive to osimertinib (a third-generation TKI). We also characterized a rare exon 21 germline variant, EGFR P848L, which transformed Ba/F3 cells and conferred resistance to multiple EGFR-targeting TKIs. Our analysis revealed that P848L (a) does not bind erlotinib; (b) is turned over less rapidly than L858R (a common tumor-derived EGFR mutation); (c) is not autophosphorylated at Tyr 1045 [the major docking site for Cbl proto-oncogene (c-Cbl) binding]; and (d) does not bind c-Cbl. Using viability assays including 300 clinically relevant targeted compounds, we observed that Ba/F3 cells transduced with EGFR P848L, S768insVGH, or L858R have very different drug-sensitivity profiles. In particular, EGFR P848L, but not L858R or S768insVGH, was sensitive to multiple Janus kinase 1/2 inhibitors. In contrast, cells driven by L858R, but not by P848L, were sensitive to multikinase MAPK/extracellular-signal-regulated kinase (ERK) kinase and ERK inhibitors including EGFR-specific TKIs. These observations suggest that continued investigation of rare TKI-resistant EGFR variants is warranted to identify optimal treatments for cancer., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Despite recent successes of precision and immunotherapies there is a persisting need for novel targeted or multi-targeted approaches in complex diseases. Through a systems pharmacology approach, including phenotypic screening, chemical and phosphoproteomics, and RNA-seq, we elucidated the targets and mechanisms underlying the differential anticancer activity of two structurally related multi-kinase inhibitors, foretinib, and cabozantinib, in lung cancer cells. Biochemical and cellular target validation using probe molecules and RNAi revealed a polypharmacology mechanism involving MEK1/2, FER, and AURKB, which were each more potently inhibited by foretinib than cabozantinib. Based on this, we developed a synergistic combination of foretinib with barasertib, a more potent AURKB inhibitor, for MYC-amplified small-cell lung cancer. This systems pharmacology approach showed that small structural changes of drugs can cumulatively, through multiple targets, result in pronounced anticancer activity differences and that detailed mechanistic understanding of polypharmacology can enable repurposing opportunities for cancers with unmet medical need., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance. Experimental Design: Mass spectrometry-based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma., Results: We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3-mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo , the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling., Conclusions: Together, our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma., (©2019 American Association for Cancer Research.)

GSK3α has been identified as a new target in the treatment of acute myeloid leukemia (AML). However, most GSK3 inhibitors lack specificity for GSK3α over GSK3β and other kinases. We have previously shown in lung cancer cells that GSK3α and to a lesser extent GSK3β are inhibited by the advanced clinical candidate tivantinib (ARQ197), which was designed as a MET inhibitor. Thus, we hypothesized that tivantinib would be an effective therapy for the treatment of AML. Here, we show that tivantinib has potent anticancer activity across several AML cell lines and primary patient cells. Tivantinib strongly induced apoptosis, differentiation and G2/M cell cycle arrest and caused less undesirable stabilization of β-catenin compared to the pan-GSK3 inhibitor LiCl. Subsequent drug combination studies identified the BCL-2 inhibitor ABT-199 to synergize with tivantinib while cytarabine combination with tivantinib was antagonistic. Interestingly, the addition of ABT-199 to tivantinib completely abrogated tivantinib induced β-catenin stabilization. Tivantinib alone, or in combination with ABT-199, downregulated anti-apoptotic MCL-1 and BCL-XL levels, which likely contribute to the observed synergy. Importantly, tivantinib as single agent or in combination with ABT-199 significantly inhibited the colony forming capacity of primary patient AML bone marrow mononuclear cells. In summary, tivantinib is a novel GSK3α/β inhibitor that potently kills AML cells and tivantinib single agent or combination therapy with ABT-199 may represent attractive new therapeutic opportunities for AML.

Resistance to androgen receptor (AR) antagonists is a significant problem in the treatment of castration-resistant prostate cancers (CRPC). Identification of the mechanisms by which CRPCs evade androgen deprivation therapies (ADT) is critical to develop novel therapeutics. We uncovered that CRPCs rely on BRD4-HOXB13 epigenetic reprogramming for androgen-independent cell proliferation. Mechanistically, BRD4, a member of the BET bromodomain family, epigenetically promotes HOXB13 expression. Consistently, genetic disruption of HOXB13 or pharmacological suppression of its mRNA and protein expression by the novel dual-activity BET bromodomain-kinase inhibitors directly correlates with rapid induction of apoptosis, potent inhibition of tumor cell proliferation and cell migration, and suppression of CRPC growth. Integrative analysis revealed that the BRD4-HOXB13 transcriptome comprises a proliferative gene network implicated in cell-cycle progression, nucleotide metabolism, and chromatin assembly. Notably, although the core HOXB13 target genes responsive to BET inhibitors (HOTBIN10) are overexpressed in metastatic cases, in ADT-treated CRPC cell lines and patient-derived circulating tumor cells (CTC) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlates with HOXB13 expression in CTCs of mCRPC patients who did not respond to abiraterone (ABR), suggesting that AURKB inhibitors could be used additionally against high-risk HOXB13-positive metastatic prostate cancers. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core proproliferative network driving ADT resistance that is targetable with potent dual-activity bromodomain-kinase inhibitors., (©2018 American Association for Cancer Research.)

Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDPK1 and AURKA was required to elicit midostaurin's cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the potent PLK1 inhibitor BI2536 elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be used for the rational design of synergistic drug combinations with high potential for clinical translation., (© 2018 Ctortecka et al.)

Targeted therapy options are currently lacking for the heterogeneous population of patients whose melanomas lack BRAF or NRAS mutations (∼35% of cases). We undertook a chemical biology screen to identify potential novel drug targets for this understudied group of tumors. Screening a panel of 8 BRAF/NRAS -WT melanoma cell lines against 240 targeted drugs identified ceritinib and trametinib as potential hits with single-agent activity. Ceritinib enhanced the efficacy of trametinib across the majority of the BRAF/NRAS -WT cell lines, and the combination showed increased cytotoxicity in both three-dimensional spheroid culture and long-term colony formation experiments. Coadministration of ceritinib and trametinib led to robust inhibition of tumor growth in an in vivo xenograft BRAF/NRAS -WT melanoma model; this was not due to ALK inhibition by ceritinib. Mechanistic studies showed the ceritinib-trametinib combination to increase suppression of MAPK and TORC1 signaling. Similar results were seen when BRAF/NRAS -WT melanoma cells were treated with a combination of trametinib and the TORC1/2 inhibitor INK128. We next used mass spectrometry-based chemical proteomics and identified known and new ceritinib targets, such as IGF1R and ACK1, respectively. Validation studies suggested that ceritinib could suppress mTORC1 signaling in the presence of trametinib through inhibition of IGF1R and/or ACK1 in a cell line-dependent manner. Together, our studies demonstrated that combining a specific inhibitor (trametinib) with a more broadly targeted agent (ceritinib) has efficacy against tumors with heterogeneous mutational profiles. Mol Cancer Ther; 17(1); 73-83. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF-mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS-mutant and KRAS-mutant cancer cell lines than vemurafenib. Using mass spectrometry-based chemical proteomics, we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS- and KRAS-mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho-CHK1, decreased CDK4 expression, and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS- and KRAS-mutant cancer cells by dabrafenib, but not vemurafenib. Combined silencing of NEK9 and CDK16 was associated with enhanced inhibition of melanoma cell proliferation. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and our studies suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

New targeted therapies are needed for advanced thyroid cancer. Our lab has shown that Src is a key mediator of tumorigenic processes in thyroid cancer. However, single-agent Src inhibitors have had limited efficacy in solid tumors. In order to more effectively target Src in the clinic, our lab has previously generated four thyroid cancer cell lines that are resistant to dasatinib through gradual dose escalation. We further tested two additional Src inhibitors and shown the dasatinib-resistant (DasRes) cells exhibit cross-resistance to saracatinib, but are sensitive to bosutinib, suggesting that unique off-targets of bosutinib play an important role in mediating sensitivity to bosutinib. To identify the kinases targeted by dasatinib and bosutinib, we utilized an unbiased compound centric chemical proteomics screen. We identified 33 kinases that were enriched in the bosutinib pull down. Using the STRING database to map protein-protein interactions of the unique bosutinib targets, we identified a signaling axis which included mTOR, FAK, and MEK. Inhibition of the mTOR, MEK, and Src/FAK nodes simultaneously was the most effective at reducing cell growth and survival. Overall, these studies have identified key mediators of Src inhibitor resistance, and show that targeting these signaling nodes are necessary for anti-tumor efficacy., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Oncogenic kinase fusions of ALK , ROS1 , RET , and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1 + lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion-positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551-63. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The "conditionally reprogrammed cells" (CRC) method, using a Rho kinase inhibitor and irradiated mouse fibroblast cells has been described for the efficient growth of cells from malignant and non-malignant samples from primary tumor and non-malignant sites. Using the CRC method, four institutions independently cultured tumor tissues from 48 non-small cell lung cancers (NSCLC, mostly from primary resected tumors) and 22 non-malignant lungs. We found that epithelial cells could be cultured from tumor and non-malignant lung. However, epithelial cells cultured from tumors had features of non-malignant respiratory epithelial cells which include: 1) among 22 mutations found in the original tumors only two mutations were found in the CRC cultures with reduced frequency (31% to 13% and 92% to 15% from original tumor and CRC culture respectively); 2) copy number variation was analyzed in 9 tumor and their CRC cultures and only diploid patterns were found in CRC cultures; 3) mRNA expression profiles were similar to those of normal respiratory epithelial cells; and 4) co-culture of tumor and non-malignant lung epithelial cells resulted in mostly non-malignant cells. We conclude that CRC method is a highly selective and useful method for the growth of non-malignant respiratory epithelial cells from tumor specimens and only occasionally do such CRC cultures contain a small subpopulation of cancer cells marked by oncogenic mutations. While our findings are restricted to resected primary NSCLC, they indicated the necessity to fully characterize all CRC cultures and the need to develop culture technology that facilitates the growth of primary lung cancers.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a promising class of targeted cancer drugs, but their individual target profiles beyond the PARP family, which could result in differential clinical use or toxicity, are unknown. Using an unbiased, mass spectrometry-based chemical proteomics approach, we generated a comparative proteome-wide target map of the four clinical PARPi, olaparib, veliparib, niraparib, and rucaparib. PARPi as a class displayed high target selectivity. However, in addition to the canonical targets PARP1, PARP2, and several of their binding partners, we also identified hexose-6-phosphate dehydrogenase (H6PD) and deoxycytidine kinase (DCK) as previously unrecognized targets of rucaparib and niraparib, respectively. Subsequent functional validation suggested that inhibition of DCK by niraparib could have detrimental effects when combined with nucleoside analog pro-drugs. H6PD silencing can cause apoptosis and further sensitize cells to PARPi, suggesting that H6PD may be, in addition to its established role in metabolic disorders, a new anticancer target., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The DNA damage response (DDR) involves a complex network of signaling events mediated by modular protein domains such as the BRCA1 C-terminal (BRCT) domain. Thus, proteins that interact with BRCT domains and are a part of the DDR constitute potential targets for sensitization to DNA-damaging chemotherapy agents. We performed a pharmacologic screen to evaluate 17 kinases, identified in a BRCT-mediated interaction network as targets to enhance platinum-based chemotherapy in lung cancer. Inhibition of mitotic kinase WEE1 was found to have the most effective response in combination with platinum compounds in lung cancer cell lines. In the BRCT-mediated interaction network, WEE1 was found in complex with PAXIP1, a protein containing six BRCT domains involved in transcription and in the cellular response to DNA damage. We show that PAXIP1 BRCT domains regulate WEE1-mediated phosphorylation of CDK1. Furthermore, ectopic expression of PAXIP1 promotes enhanced caspase-3-mediated apoptosis in cells treated with WEE1 inhibitor AZD1775 (formerly, MK-1775) and cisplatin compared with cells treated with AZD1775 alone. Cell lines and patient-derived xenograft models expressing both PAXIP1 and WEE1 exhibited synergistic effects of AZD1775 and cisplatin. In summary, PAXIP1 is involved in sensitizing lung cancer cells to the WEE1 inhibitor AZD1775 in combination with platinum-based treatment. We propose that WEE1 and PAXIP1 levels may be used as mechanism-based biomarkers of response when WEE1 inhibitor AZD1775 is combined with DNA-damaging agents. Mol Cancer Ther; 15(7); 1669-81. ©2016 AACR., (©2016 American Association for Cancer Research.)

To overcome hurdles in identifying key kinases in small cell lung cancer (SCLC), we integrated a target-agnostic phenotypic screen of kinase inhibitors with target identification using activity-based protein profiling (ABPP) in which a desthiobiotin-ATP probe was used. We screened 21 SCLC cell lines with known c-MYC amplification status for alterations in viability using a chemical library of 235 small-molecule kinase inhibitors. One screen hit compound was interrogated with ABPP, and, through this approach, we reidentified Aurora kinase B as a critical kinase in MYC-amplified SCLC cells. We next extended the platform to a second compound that had activity in SCLC cell lines lacking c-MYC amplification and identified TANK-binding kinase 1, a kinase that affects cell viability, polo-like kinase-1 signaling, G2-M arrest, and apoptosis in SCLC cells lacking MYC amplification. These results demonstrate that phenotypic screening combined with ABPP can identify key disease drivers, suggesting that this approach, which combines new chemical probes and disease cell screens, has the potential to identify other important targets in other cancer types. Mol Cancer Ther; 15(2); 334-42. ©2016 AACR., (©2016 American Association for Cancer Research.)

Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

DDR2 mutations occur in approximately 4% of lung squamous cell cancer (SCC) where the tyrosine kinase inhibitor dasatinib has emerged as a new therapeutic option. We found that ERK and AKT phosphorylation was weakly inhibited by dasatinib in DDR2-mutant lung SCC cells, suggesting that dasatinib inhibits survival signals distinct from other oncogenic receptor tyrosine kinases (RTK) and/or compensatory signals exist that dampen dasatinib activity. To gain better insight into dasatinib's action in these cells, we assessed altered global tyrosine phosphorylation (pY) after dasatinib exposure using a mass spectrometry-based quantitative phosphoproteomics approach. Overlaying protein-protein interaction relationships upon this dasatinib-regulated pY network revealed decreased phosphorylation of Src family kinases and their targets. Conversely, dasatinib enhanced tyrosine phosphorylation in a panel of RTK and their signaling adaptor complexes, including EGFR, MET/GAB1, and IGF1R/IRS2, implicating a RTK-driven adaptive response associated with dasatinib. To address the significance of this observation, these results were further integrated with results from a small-molecule chemical library screen. We found that dasatinib combined with MET and insulin-like growth factor receptor (IGF1R) inhibitors had a synergistic effect, and ligand stimulation of EGFR and MET rescued DDR2-mutant lung SCC cells from dasatinib-induced loss of cell viability. Importantly, we observed high levels of tyrosine-phosphorylated EGFR and MET in a panel of human lung SCC tissues harboring DDR2 mutations. Our results highlight potential RTK-driven adaptive-resistant mechanisms upon DDR2 targeting, and they suggest new, rationale cotargeting strategies for DDR2-mutant lung SCC., (©2014 American Association for Cancer Research.)

A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. While these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib), and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune, and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes, and signaling pathways that are simultaneously engaged by multitargeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments., (©2014 American Association for Cancer Research.)

Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.

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.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is in part driven by the tyrosine kinase bcr-abl, but imatinib does not produce long-term remission. Therefore, second-generation ABL inhibitors are currently in clinical investigation. Considering different target specificities and the pronounced genetic heterogeneity of Ph+ ALL, which contributes to the aggressiveness of the disease, drug candidates should be evaluated with regard to their effects on the entire Ph+ ALL-specific signaling network. Here, we applied an integrated experimental and computational approach that allowed us to estimate the differential impact of the bcr-abl inhibitors nilotinib, dasatinib, Bosutinib and Bafetinib. First, we determined drug-protein interactions in Ph+ ALL cell lines by chemical proteomics. We then mapped those interactions along with known genetic lesions onto public protein-protein interactions. Computation of global scores through correlation of target affinity, network topology, and distance to disease-relevant nodes assigned the highest impact to dasatinib, which was subsequently confirmed by proliferation assays. In future, combination of patient-specific genomic information with detailed drug target knowledge and network-based computational analysis should allow for an accurate and individualized prediction of therapy.

TANK-binding kinase 1 (TBK1) has emerged as a novel therapeutic target for unspecified subset of lung cancers. TBK1 reportedly mediates prosurvival signaling by activating NF-κB and AKT. However, we observed that TBK1 knockdown also decreased viability of cells expressing constitutively active NF-κB and interferon regulatory factor 3. Basal phospho-AKT level was not reduced after TBK1 knockdown in TBK1-sensitive lung cancer cells, implicating that TBK1 mediates unknown survival mechanisms. To gain better insight into TBK1 survival signaling, we searched for altered phosphoproteins using mass spectrometry following RNAi-mediated TBK1 knockdown. In total, we identified 2,080 phosphoproteins (4,621 peptides), of which 385 proteins (477 peptides) were affected after TBK1 knockdown. A view of the altered network identified a central role of Polo-like kinase 1 (PLK1) and known PLK1 targets. We found that TBK1 directly phosphorylated PLK1 in vitro. TBK1 phosphorylation was induced at mitosis, and loss of TBK1 impaired mitotic phosphorylation of PLK1 in TBK1-sensitive lung cancer cells. Furthermore, lung cancer cell sensitivity to TBK1 was highly correlated with sensitivity to pharmacological PLK inhibition. We additionally found that TBK1 knockdown decreased metadherin phosphorylation at Ser-568. Metadherin was associated with poor outcome in lung cancer, and loss of metadherin caused growth inhibition and apoptosis in TBK1-sensitive lung cancer cells. These results collectively revealed TBK1 as a mitosis regulator through activation of PLK1 and also suggested metadherin as a putative TBK1 downstream effector involved in lung cancer cell survival.

Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.

Systemic mastocytosis (SM) either presents as a malignant neoplasm with short survival or as an indolent disease with normal life expectancy. In both instances, neoplastic mast cells (MCs) harbor D816V-mutated KIT, suggesting that additional oncogenic mechanisms are involved in malignant transformation. We here describe that Lyn and Btk are phosphorylated in a KIT-independent manner in neoplastic MCs in advanced SM and in the MC leukemia cell line HMC-1. Lyn and Btk activation was not only detected in KIT D816V-positive HMC-1.2 cells, but also in the KIT D816V-negative HMC-1.1 subclone. Moreover, KIT D816V did not induce Lyn/Btk activation in Ba/F3 cells, and deactivation of KIT D816V by midostaurin did not alter Lyn/Btk activation. siRNAs against Btk and Lyn were found to block survival in neoplastic MCs and to cooperate with midostaurin in producing growth inhibition. Growth inhibitory effects were also obtained with 2 targeted drugs, dasatinib which blocks KIT, Lyn, and Btk activation in MCs, and bosutinib, a drug that deactivates Lyn and Btk without blocking KIT activity. Together, KIT-independent signaling via Lyn/Btk contributes to growth of neoplastic MCs in advanced SM. Dasatinib and bosutinib disrupt Lyn/Btk-driven oncogenic signaling in neoplastic MC, which may have clinical implications and explain synergistic drug interactions.

Prediction of drug action in human cells is a major challenge in biomedical research. Additionally, there is strong interest in finding new applications for approved drugs and identifying potential side effects. We present a computational strategy to predict mechanisms, risks and potential new domains of drug treatment on the basis of target profiles acquired through chemical proteomics. Functional protein-protein interaction networks that share one biological function are constructed and their crosstalk with the drug is scored regarding function disruption. We apply this procedure to the target profile of the second-generation BCR-ABL inhibitor bafetinib which is in development for the treatment of imatinib-resistant chronic myeloid leukemia. Beside the well known effect on apoptosis, we propose potential treatment of lung cancer and IGF1R expressing blast crisis.