Guerra, S.L., et al. A Deregulated HOX Gene Axis Confers an Epigenetic Vulnerability in KRAS-Mutant Lung Cancers. Cancer Cell (2020).Abstract
While KRAS mutations are common in non-small cell lung cancer (NSCLC), effective treatments are lacking. Here, we report that half of KRAS-mutant NSCLCs aberrantly express the homeobox protein HOXC10, largely due to unappreciated defects in PRC2, which confers sensitivity to combined BET/MEK inhibitors in xenograft and PDX models. Efficacy of the combination is dependent on suppression of HOXC10 by BET inhibitors. We further show that HOXC10 regulates the expression of pre-replication complex (pre-RC) proteins in sensitive tumors. Accordingly, BET/MEK inhibitors suppress pre-RC proteins in cycling cells, triggering stalled replication, DNA damage, and death. These studies reveal a promising therapeutic strategy for KRAS-mutant NSCLCs, identify a predictive biomarker of response, and define a subset of NSCLCs with a targetable epigenetic vulnerability.
Färkkilä, A., et al. Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer. Nat Commun 11, 1, 1459 (2020).Abstract
Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.
Gaglia, G., et al. HSF1 phase transition mediates stress adaptation and cell fate decisions. Nat Cell Biol (2020).Abstract
Under proteotoxic stress, some cells survive whereas others die. The mechanisms governing this heterogeneity in cell fate remain unknown. Here we report that condensation and phase transition of heat-shock factor 1 (HSF1), a transcriptional regulator of chaperones, is integral to cell-fate decisions underlying survival or death. During stress, HSF1 drives chaperone expression but also accumulates separately in nuclear stress bodies called foci. Foci formation has been regarded as a marker of cells actively upregulating chaperones. Using multiplexed tissue imaging, we observed HSF1 foci in human tumours. Paradoxically, their presence inversely correlated with chaperone expression. By live-cell microscopy and single-cell analysis, we found that foci dissolution rather than formation promoted HSF1 activity and cell survival. During prolonged stress, the biophysical properties of HSF1 foci changed; small, fluid condensates enlarged into indissoluble gel-like arrangements with immobilized HSF1. Chaperone gene induction was reduced in such cells, which were prone to apoptosis. Quantitative analysis suggests that survival under stress results from competition between concurrent but opposing mechanisms. Foci may serve as sensors that tune cytoprotective responses, balancing rapid transient responses and irreversible outcomes.
Maertens, O., et al. MAPK Pathway Suppression Unmasks Latent DNA Repair Defects and Confers a Chemical Synthetic Vulnerability in -, and -Mutant Melanomas. Cancer Discov 9, 4, 526-545 (2019).Abstract
Although the majority of -mutant melanomas respond to BRAF/MEK inhibitors, these agents are not typically curative. Moreover, they are largely ineffective in - and -mutant tumors. Here we report that genetic and chemical suppression of HDAC3 potently cooperates with MAPK pathway inhibitors in all three RAS pathway-driven tumors. Specifically, we show that entinostat dramatically enhances tumor regression when combined with BRAF/MEK inhibitors, in both models that are sensitive or relatively resistant to these agents. Interestingly, expression predicts responsiveness and marks tumors with latent defects in DNA repair. BRAF/MEK inhibitors enhance these defects by suppressing homologous recombination genes, inducing a BRCA-like state; however, addition of entinostat triggers the concomitant suppression of nonhomologous end-joining genes, resulting in a chemical synthetic lethality caused by excessive DNA damage. Together, these studies identify melanomas with latent DNA repair defects, describe a promising drug combination that capitalizes on these defects, and reveal a tractable therapeutic biomarker. SIGNIFICANCE: BRAF/MEK inhibitors are not typically curative in -mutant melanomas and are ineffective in - and -mutant tumors. We show that HDAC inhibitors dramatically enhance the efficacy of BRAF/MEK inhibitors in sensitive and insensitive RAS pathway-driven melanomas by coordinately suppressing two DNA repair pathways, and identify a clinical biomarker that predicts responsiveness...
Pantelidou, C., et al. PARP Inhibitor Efficacy Depends on CD8 T-cell Recruitment via Intratumoral STING Pathway Activation in BRCA-Deficient Models of Triple-Negative Breast Cancer. Cancer Discov 9, 6, 722-737 (2019).Abstract
Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8 T-cell infiltration and activation , and that CD8 T-cell depletion severely compromises antitumor efficacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared with HR-proficient TNBC cells and models. CRISPR-mediated knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T-cell infiltration . These findings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. SIGNIFICANCE: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8 T-cell recruitment and antitumor efficacy. The data provide insight into the mechanism of action of PARP inhibitors in -associated breast cancer..
Palmer, A.C., Chidley, C. & Sorger, P.K. A curative combination cancer therapy achieves high fractional cell killing through low cross-resistance and drug additivity. Elife 8, (2019).Abstract
Curative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here, we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R-CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R-CHOP exhibit very low cross-resistance but not synergistic interaction: together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50 year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.
Chopra, S.S., et al. Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells. Cell Syst (2019).Abstract
Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.
Doebele, R.C. Acquired Resistance Is Oncogene and Drug Agnostic. Cancer Cell 36, 4, 347-349 (2019).Abstract
Recent approvals of TRK inhibitors have demonstrated the success of a tumor agnostic approach to oncogene-targeted therapy across cancers. Collective data from acquired resistance studies suggest that resistance mechanisms, which include both kinase domain mutations and bypass signaling via RTK-RAS-RAF-MAPK pathways, frequently recur regardless of tumor type, oncogene, and drug.
Togami, K., et al. DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance. J Clin Invest (2019).Abstract
The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.
Rajkumar-Calkins, A.S., et al. Functional profiling of nucleotide Excision repair in breast cancer. DNA Repair (Amst) 82, 102697 (2019).Abstract
Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.
Labbé, D.P., et al. High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program. Nat Commun 10, 1, 4358 (2019).Abstract
Systemic metabolic alterations associated with increased consumption of saturated fat and obesity are linked with increased risk of prostate cancer progression and mortality, but the molecular underpinnings of this association are poorly understood. Here, we demonstrate in a murine prostate cancer model, that high-fat diet (HFD) enhances the MYC transcriptional program through metabolic alterations that favour histone H4K20 hypomethylation at the promoter regions of MYC regulated genes, leading to increased cellular proliferation and tumour burden. Saturated fat intake (SFI) is also associated with an enhanced MYC transcriptional signature in prostate cancer patients. The SFI-induced MYC signature independently predicts prostate cancer progression and death. Finally, switching from a high-fat to a low-fat diet, attenuates the MYC transcriptional program in mice. Our findings suggest that in primary prostate cancer, dietary SFI contributes to tumour progression by mimicking MYC over expression, setting the stage for therapeutic approaches involving changes to the diet.
Zhang, W.C., et al. miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma. Nat Metab 1, 4, 460-474 (2019).Abstract
Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse, however there are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. With miRNA-seq analysis we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and mutated lung cancer cells. Whole transcriptome analysis of single-cell derived clones reveals a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived three-dimensional (3D) structures. This link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.
Guièze, R., et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell 36, 4, 369-384.e13 (2019).Abstract
Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.
Ding, L., et al. Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ. Nat Commun 10, 1, 4182 (2019).Abstract
Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63TCF7 myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63TCF7 myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression.
Stover, E.H., et al. Pooled Genomic Screens Identify Anti-apoptotic Genes as Targetable Mediators of Chemotherapy Resistance in Ovarian Cancer. Mol Cancer Res (2019).Abstract
High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including (BCL-XL) and (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.
LaFleur, M.W., et al. PTPN2 regulates the generation of exhausted CD8 T cell subpopulations and restrains tumor immunity. Nat Immunol 20, 10, 1335-1347 (2019).Abstract
CD8 T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6 progenitor exhausted and Tim-3 terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8 T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3 cells without altering Slamf6 numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8 T cells enhanced Tim-3 anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3CD8 T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.
Du, Z., et al. Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nat Protoc 14, 10, 2900-2930 (2019).Abstract
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
Ron-Harel, N., et al. T Cell Activation Depends on Extracellular Alanine. Cell Rep 28, 12, 3011-3021.e4 (2019).Abstract
T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation.
Niepel, M., et al. A Multi-center Study on the Reproducibility of Drug-Response Assays in Mammalian Cell Lines. Cell Syst 9, 1, 35-48.e5 (2019).Abstract
Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in practices that generate findable, accessible, interoperable, and reusable (FAIR) data. Multiple papers have identified specific examples of irreproducibility, but practical ways to make data more reproducible have not been widely studied. Here, five research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cellular networks, and patient stratification. While many experimental and computational factors impact intra- and inter-center reproducibility, the factors most difficult to identify and control are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways to identify such context-sensitive factors, thereby improving both the theory and practice of reproducible cell-based assays.
Hafner, M., et al. Multiomics Profiling Establishes the Polypharmacology of FDA-Approved CDK4/6 Inhibitors and the Potential for Differential Clinical Activity. Cell Chem Biol (2019).Abstract
The target profiles of many drugs are established early in their development and are not systematically revisited at the time of FDA approval. Thus, it is often unclear whether therapeutics with the same nominal targets but different chemical structures are functionally equivalent. In this paper we use five different phenotypic and biochemical assays to compare approved inhibitors of cyclin-dependent kinases 4/6-collectively regarded as breakthroughs in the treatment of hormone receptor-positive breast cancer. We find that transcriptional, proteomic, and phenotypic changes induced by palbociclib, ribociclib, and abemaciclib differ significantly; abemaciclib in particular has advantageous activities partially overlapping those of alvocidib, an older polyselective CDK inhibitor. In cells and mice, abemaciclib inhibits kinases other than CDK4/6 including CDK2/cyclin A/E-implicated in resistance to CDK4/6 inhibition-and CDK1/cyclin B. The multifaceted experimental and computational approaches described here therefore uncover underappreciated differences in CDK4/6 inhibitor activities with potential importance in treating human patients.