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Supplemental figure legends 1-8, Supplementary tables 1-5, and Supplementary methods

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Supplemental Figures 1-8

PDOTS Movie 2

Application of Bliss independence to test for synergy effect of TKI combination treatment in vivo

PDF file - 1334KB, S1. IC50 values of TBK1 inhibitors Compound number 5, Compound number 4 and Compound number 3 from the selected PDAC, colorectal cancer and NSCLC cancer cell lines using are shown. Table 1S. List of shRNAs Targeting TBK1 Table 2S. RAS Gene Signature Score Correlation with Sensitivity to the TBK1i (Compound #1) and Meki (PD0325901) in NSCLC Cell Lines Table 3S. List of Cell Lines from 6-Cell Line Panels that were Used in TBK1 Inhibitor Screening in the 2-Dimensional Cell Viability Assay (NSCLC and Colorectal cancer panels) Table 4S. List of Cell Lines from 6-Cell Line Panels that were Used in TBK1 Inhibitor Screening in the 2-Dimensional Cell Viability Assay (Ovarian cancer, GBM and Lymphoma panels).

In vitro kinase inhibitory activity of Compound 1

Table 1 compound list

We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b–restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non–small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro. The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Table 3 SCCHN clinical info

Suppl. Fig. 1-6

Table S3 shows list of KRAS WT; LKB1 WT and KRAS WT; LKB1 Mut lung adenocarcinoma cell lines related with supplementary figure S1.

Supplementary Figures S1-3; Supplementary Tables S1-3

Table 2 sgRNA genes and sequences

The supplementary file shows Supplementary figure S1-S4 and their legends, and the legends for Supplementary table S1-S4.

Device loading, media addition, media extraction

Supplementary Figure Legends and Figures

PDOTS Movie

Detailed description of methodology and reagents

Synergism of prexasertib and olaparib in ovarian cancer cell lines

Next generation sequencing copy number plots for Case #2

Summary of EGFR inhibitor resistant patient-derived xenografts.

Supplementary Figure Legends

Antibodies used in the study

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non–small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3–DXd is an antibody–drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3–DXd across a series of EGFR inhibitor–resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3–DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3–DXd. The combination of osimertinib and HER3–DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients.Significance:EGFR inhibition leads to increased HER3 membrane expression and promotes HER3–DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3–DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18

Purpose:Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy.Experimental Design:We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors.Results:Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti–PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti–PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23–3.16; P = 0.005].Conclusions:Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.

Allelic Frequencies in PC-9 vs PC-9 T790M/C797S cells.

Antibodies, compounds, oligonucleotides, and other reagents used in the study

Figure S1

Characterization of high-grade serous ovarian cancer models

Purpose:PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance.Experimental Design:Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed.Results:Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability.Conclusions:Prexasertib exhibits monotherapy activity in PARP inhibitor–resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.

Table S4: Somatic Sequence Alterations in parental tumor and xenograft DF101

Table S5: Somatic Sequence Alterations in parental tumor and xenograft DF149

Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease.Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden.Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers.Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263–73. ©2016 AACR.

Table S2: Array CGH characterization of PDX models

Supplementary Figures and legends

Supplementary Methods and Legends

Table S1

cDNA clones representing genes whose expression is modulated by treatment with mitogens and tumor promoters were isolated and characterized. TPA-S1 corresponds to an mRNA species whose abundance was increased markedly within 1 h of exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), and TPA-R1 represents an mRNA that was decreased in TPA-treated cells. The induction of TPA-S1 was blocked by actinomycin D but was not affected by cycloheximide, and it was specific for phorbol esters with tumor-promoting activity. The role of protein kinase C in the induction of TPA-S1 is supported by the following lines of evidence. (i) Agents that activated protein kinase C (TPA, platelet-derived growth factor, and diacylglycerol) also increased TPA-S1 mRNA levels. (ii) A potent PKC inhibitor blocked the induction of TPA-S1. (iii) Down-regulation of PKC activity, by treatment of cells with TPA for 24 h, resulted in a loss of responsiveness to TPA-S1 induction by subsequent TPA treatment. DNA sequence analysis of TPA-S1 predicts a cysteine-rich, secreted protein with a molecular weight of 22.6 X 10(3) that exhibits homology with sequences representing a protein with human erythroid-potentiating activity and protease inhibitory activity.

Summary We have generated a series of rat fibroblast cell linesthat stably overexpress a full-length cDNA encoding the β1 form of protein kinase C (PKC). These cell lines contain a 20-to 53-fold increase in PKC activity and exhibit dramatically enhanced morphologic changes following exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). They grow to a high saturation density in monolayer cultures and, when maintained at postconfluence, develop small, dense foci. In contrast to control cells, which display complete anchorage dependence, PKC-overproducing cells form small colonies in soft agar in the absence of TPA and large colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer multiple growth abnormalities in rat fibroblasts. These results provide direct evidence that PKC plays a critical role in growth control and that it mediates several of the cellular effects of the phorbol ester tumor promoters. They also suggest that the activation of PKC may be of central importance in the process of multistage carcinogenesis.

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

RNA sequences homologous to the Long Terminal Repeat (LTR) sequence of Moloney Murine Leukemia Virus proviral DNA are expressed in murine squamous cell carcinomas of the skin induced by chemical carcinogens. These transcripts range in size from 8.2 to less than 2.4 kb but their size profile varies between individual tumors. These RNAs are not detected in the poly A+ RNA fraction obtained from the epidermis of control mice or carcinogen induced skin papillomas. The poly A+ RNAs from the livers and spleens of some of the mice with skin carcinomas also revealed LTR related sequences, whereas these RNAs were not detected in the livers and spleens of control mice or of carcinogen-treated mice that did not develop carcinomas. Thus, chemical carcinogenesis in mouse skin is associated with constitutive expression of endogenous retrovirus related sequences in the carcinomas as well as in certain apparently normal host tissues.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has highly pleiotropic effects on cells in culture and on tissues in vivo, including effects on protein kinase C (PKC) activation and gene expression. In order to determine the mechanism of activation of gene transcription by TPA, DNA sequences whose transcription is modulated in cells undergoing a mitogenic response to TPA were isolated by differential screening of a cDNA library from TPA-treated cells. TPA-S1 corresponds to an mRNA species whose abundance is increased within 1 hr of exposure of quiescent C3H 10T1/2 mouse embryo fibroblasts. TPA-R1 corresponds to an mRNA species whose abundance is decreased in TPA-treated cells. The induction of TPA-S1 is blocked by actinomycin D and is specific for phorbol esters with tumor-promoting activity. The transcription of this sequence is not induced by cycloheximide, nor is there an enhancement of the TPA response. Several lines of evidence demonstrate that PKC activation plays a critical role in the regulation of TPA-S1 expression. The nucleotide and predicted amino acid sequence of TPA-S1 exhibits homology with sequences representing a peptide with erythroid-potentiating activity, a metalloproteinase inhibitor protein, and a murine protein with beta-interferon-like activity. The role of TPA-S1 in tumor promotion is suggested by the expression of this sequence in mouse skin carcinomas induced by dimethyl-benzanthracene-TPA treatment, but not in papillomas or in control tissue. The consideration of signal transduction pathways may be useful in the design of short-term risk assessment assays for agents that act as tumor promoters. Images FIGURE 2. FIGURE 3. FIGURE 4.

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.

Previous reports on retroviral vectors have shown them to be useful for transferring genes into animal cells. Genes placed under the retroviral long terminal repeat (LTR) act as dominant loci in recipient cells and can permanently alter their genotype and phenotype. Previous reports have shown that recombinant retroviruses containing genomic sequences with both introns and exons display a high frequency of deletion and abnormal kinetics of splicing of intron sequences. We report here our findings when a 2.9-kb fragment containing the entire human insulin gene was inserted into a Moloney-derived retroviral vector in the same transcriptional orientation as the LTRs. RNA transcripts synthesized in cells containing such constructs remain unspliced, as assessed by both RNA blot analysis and S1 mapping. Ten subclones derived following viral passage showed no splicing, and failure to splice was observed regardless of cell type or species of origin, or number of viral passages. Thus, genomic sequences containing introns when situated within the context of a retroviral transcript do not in all instances exhibit expected kinetics of splicing.

Research on growth factors, growth factor receptors and signal transduction pathways have provided an exciting conceptual framework for understanding multistage carcinogenesis1. Fig. 1 displays in schematic form how certain extracellular growth factors are perceived by cellular receptors, which are often located at the cell surface, and how the occupancy of these receptors leads to a cascade of signal transduction, through the cytoplasm and eventually into the nucleus, thus altering patterns of gene expression. This figure also emphasizes the central role that a series of protein kinase enzymes plays in several pathways of signal transduction. A general theme that has emerged is that the proto-oncogenes represent a subset of genes that normally code for components in these pathways of signal transduction. Alterations in the structure and function of these proto-oncogenes can convert them to “activated” oncogenes, which cause aberrations in signal transduction and thus disrupt normal growth, differentiation and inter-cellular coordination.

We describe the construction and properties of a retroviral expression vector, designated pMV-7, designed to transfer unselected cDNAs and produce their encoded proteins in recipient cells. The vector is flanked by the long terminal repeats (LTRs) of the Moloney murine sarcoma virus (MSV) and contains the selectable drug resistance gene neo under the regulation of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Unique Eco RI and Hind III sites facilitate the introduction of sequences whose transcription is regulated by the 5' LTR. We have inserted cDNAs encoding: (i) the human lymphocyte antigen T4, (ii) the human lymphocyte antigen T8, and (iii) the murine hypoxanthine-guanine phosphoribosyl transferase (HPRT), into the pMV-7 vector. These constructions were used to transduce recipient cells to the neo+ phenotype. In each case, functional assays demonstrated that 65-92% of the neo+ clones produced the appropriate protein encoded by its corresponding cDNA. These clones were characterized further by analyzing the expression of vector-regulated transcripts. The neo+T4+ clones expressed a single full-length LTR-to-LTR transcript as detected by a T4 probe. The neo+T8+ clones, however, expressed both a full-length LTR-to-LTR transcript and an additional smaller transcript as detected by a T8 probe. This smaller transcript probably resulted from the utilization of cryptic signals which control 3' RNA processing. Furthermore, all of the neo+ clones expressed a transcript that initiated from the tk promoter, contained the neo gene, and used polyadenylation signals provided by the 3' LTR. Thus, the pMV-7 vector is capable of high-efficiency transfer and high-frequency expression of the cDNA-encoded protein.