No abstract

Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic mechanism of action, we systematically analyzed prostate cancer-specific CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo anti-tumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of co-stimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development novel optimized immune receptors.

Summary 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 utility 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.

Several selective CDK4/6 inhibitors are in clinical trials for non-small cell lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential cellular activity between palbociclib and the structurally related ribociclib (LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based chemoproteomics approach in H157 cells and primary tumor samples, we here report distinct proteome-wide target profiles of these two drug candidates in LUSQ, which encompass novel protein and, for palbociclib only, lipid kinases. In addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. Palbociclib interacted with several kinases not targeted by ribociclib, such as casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib engaged several lipid kinases, most notably PIK3CD and PIP4K2A/B/C. Accordingly, we observed modulation of autophagy and inhibition of AKT signaling by palbociclib, but not ribociclib.

Tivantinib has been described as a potent and highly selective inhibitor of the receptor tyrosine kinase c-MET and is currently in advanced clinical development for several cancers including non-small cell lung cancer (NSCLC). However, recent studies suggest that tivantinib’s anticancer properties are unrelated to c-MET inhibition. Consistently, in determining tivantinib’s activity profile in a broad panel of NSCLC cell lines, we found that, in contrast to several more potent c-MET inhibitors, tivantinib reduces cell viability across most of these cell lines. Applying an unbiased, mass-spectrometry-based, chemical proteomics approach, we identified glycogen synthase kinase 3 (GSK3) alpha and beta as novel tivantinib targets. Subsequent validation showed that tivantinib displayed higher potency for GSK3α than for GSK3β and that pharmacological inhibition or simultaneous siRNA-mediated loss of GSK3α and GSK3β caused apoptosis. In summary, GSK3α and GSK3β are new kinase targets of tivantinib that play an important role in its cellular mechanism-of-action in NSCLC.

Targeted drugs are effective when directly inhibiting strong disease drivers, but only a small fraction of diseases feature defined actionable drivers. Alternatively, network-based approaches can uncover new therapeutic opportunities. Applying an integrated phenotypic screening, chemical and phosphoproteomics strategy, we here describe the ALK inhibitor ceritinib to have activity across several ALK-negative lung cancer cell lines and identify new targets and network-wide signaling effects. Combining pharmacological inhibitors and RNA interference revealed a polypharmacology mechanism involving the non-canonical targets IGF1R, FAK1 and RSK1/2. Mutating the downstream signaling hub YB1 protected cells from ceritinib. Consistent with YB1 signaling being known to cause taxol resistance, ceritinib combination with paclitaxel displayed strong synergy, particularly in cells expressing high FAK autophosphorylation, which we show to be prevalent in lung cancer. Together, we present a systems chemical biology platform for elucidating multi-kinase inhibitor polypharmacology mechanisms, subsequent design of synergistic drug combinations, and identification of mechanistic biomarker candidates.

Resistance to Androgen receptor (AR) antagonists is a significant problem in the treatment of Castration resistant prostate cancers (CRPCs). 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 or pharmacological suppression of HOXB13 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, inhibits cell migration and suppresses 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, while 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 (CTCs) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlate 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 PCs. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core pro-proliferative network driving ADT resistance that is targetable with potent dual activity bromodomain-kinase inhibitors.

A key goal of cancer systems biology is to use big data to elucidate the molecular networks by which cancer develops. However, to date there has been no systematic evaluation of how far these efforts have progressed. In this Analysis, we survey six major systems biology approaches for mapping and modelling cancer pathways with attention to how well their resulting network maps cover and enhance current knowledge. Our sample of 2,070 systems biology maps captures all literature-curated cancer pathways with significant enrichment, although the strong tendency is for these maps to recover isolated mechanisms rather than entire integrated processes. Systems biology maps also identify previously underappreciated functions, such as a potential role for human papillomavirus-induced chromosomal alterations in ovarian tumorigenesis, and they add new genes to known cancer pathways, such as those related to metabolism, Hippo signalling and immunity. Notably, we find that many cancer networks have been provided only in journal figures and not for programmatic access, underscoring the need to deposit network maps in community databases to ensure they can be readily accessed. Finally, few of these findings have yet been clinically translated, leaving ample opportunity for future translational studies. Periodic surveys of