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Individual tumor growth curves; IT antibody + IT agonists

Legend for Supplementary Tables:1,2,3,4

Supplemental Contents and Legends

Suppl. Table 1. Binding mode of Debio 0617B to ABL. Suppl. Table 2. Physico-chemical and early ADME profile of Debio 0617B. Suppl. Table 3. Debio 0617B activity on a panel of 21 cancer cell lines. Suppl. Table 4. PK/PD Profile of Debio 0617B in mice with A549 tumors.

Suppl. Fig. 1 Characterization for pSTAT3 activation in cancer cell lines responding to Debio 0617B in vitro by Western blot analysis.Suppl. Fig. 2 A-C. Efficacy studies with Debio 0617B in A549, A2058 and A431 models: body weight loss/gain (%). Suppl. Fig. 3A. Histopathology of primary tumors in the 4T1 mammary tumor model. 3B. Efficacy of Debio 0617B in the 4T1 neoadjuvant mammary tumor model: body weight loss/gain (%).

Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment—including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs—can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint–modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676–84. ©2017 AACR.

Legend for Supplementary Figures:1A,1B,1C,1D 2A,2B,2C;3A,3B

High-risk human papillomavirus (HPV)–associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV− SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen–specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327–39. ©2018 AACR.

Sequential treatment of vaccine and immune checkpoint modulation

Fig. S7: HPV-specific T cells in the blood after vaccine immunotherapy. Fig. S8: Immune correlates for treatment resistance.

Supplementary Methods

Supplementary Table 4 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Supplementary Figures 1-8 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Supplementary Table 5 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Supplementary Tables 1-3 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Supplementary Methods and Materials from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Background: Hepatocellular carcinoma has high recurrence rates after surgery; however, there are no approved standard-of-care neoadjuvant or adjuvant therapies. Immunotherapy has been shown to improve survival in advanced hepatocellular carcinoma; we therefore aimed to evaluate the safety and tolerability of perioperative immunotherapy in resectable hepatocellular carcinoma. Methods: In this single-centre, randomised, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma were randomly assigned (1:1) to receive 240 mg of nivolumab intravenously every 2 weeks (for up to three doses before surgery at 6 weeks) followed in the adjuvant phase by 480 mg of nivolumab intravenously every 4 weeks for 2 years, or 240 mg of nivolumab intravenously every 2 weeks (for up to three doses before surgery) plus one dose of 1 mg/kg of ipilimumab intravenously concurrently with the first preoperative dose of nivolumab, followed in the adjuvant phase by 480 mg of nivolumab intravenously every 4 weeks for up to 2 years plus 1 mg/kg of ipilimumab intravenously every 6 weeks for up to four cycles. Patients were randomly assigned to the treatment groups by use of block randomisation with a random block size. The primary endpoint was the safety and tolerability of nivolumab with or without ipilimumab. Secondary endpoints were the proportion of patients with an overall response, time to progression, and progression-free survival. This trial is registered with ClinicalTrials.gov (NCT03222076) and is completed. Findings: Between Oct 30, 2017, and Dec 3, 2019, 30 patients were enrolled and 27 were randomly assigned: 13 to nivolumab and 14 to nivolumab plus ipilimumab. Grade 3–4 adverse events were higher with nivolumab plus ipilimumab (six [43%] of 14 patients) than with nivolumab alone (three [23%] of 13). The most common treatment-related adverse events of any grade were increased alanine aminotransferase (three [23%] of 13 patients on nivolumab vs seven [50%] of 14 patients on nivolumab plus ipilimumab) and increased aspartate aminotransferase (three [23%] vs seven [50%]). No patients in either group had their surgery delayed due to grade 3 or worse adverse events. Seven of 27 patients had surgical cancellations, but none was due to treatment-related adverse events. Estimated median progression-free survival was 9·4 months (95% CI 1·47–not estimable [NE]) with nivolumab and 19·53 months (2·33–NE) with nivolumab plus ipilimumab (hazard ratio [HR] 0·99, 95% CI 0·31–2·54); median time to progression was 9·4 months (95% CI 1·47–NE) in the nivolumab group and 19·53 months (2·33–NE) in the nivolumab plus ipilimumab group (HR 0·89, 95% CI 0·31–2·54). In an exploratory analysis, three (23%) of 13 patients had an overall response with nivolumab monotherapy, versus none with nivolumab plus ipilimumab. Three (33%) of nine patients had a major pathological response (ie, ≥70% necrosis in the resected tumour area) with nivolumab monotherapy compared with three (27%) of 11 with nivolumab plus ipilimumab. Interpretation: Perioperative nivolumab alone and nivolumab plus ipilimumab appears to be safe and feasible in patients with resectable hepatocellular carcinoma. Our findings support further studies of immunotherapy in the perioperative setting in hepatocellular carcinoma. Funding: Bristol Myers Squibb and the US National Institutes of Health.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer and commonly arises in chronically UV-exposed skin or chronic wounds. Since UV exposure and chronic wounds are the two most prominent environmental factors that lead to cuSCC initiation, we undertook this study to test whether more acute molecular responses to UV and wounding overlapped with molecular signatures of cuSCC. We reasoned that transcriptional signatures in common between acutely UV-exposed skin, wounded skin, and cuSCC tumors, might enable us to identify important pathways contributing to cuSCC. We performed transcriptomic analysis on acutely UV-exposed human skin and integrated those findings with datasets from wounded skin and our transcriptomic data on cuSCC using functional pair analysis, GSEA, and pathway analysis. Integrated analyses revealed significant overlap between these three datasets, thus highlighting deep molecular similarities these biological processes, and we identified Oncostatin M (OSM) as a potential common upstream driver. Expression of OSM and its downstream targets correlated with poorer overall survival in head and neck SCC patients. In vitro, OSM promoted invasiveness of keratinocytes and cuSCC cells and suppressed apoptosis of irradiated keratinocytes. Together, these results support the concept of using an integrated, biologically-informed approach to identify potential promoters of tumorigenesis.

Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment—including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs—can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint–modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676–84. ©2017 AACR.

Background The US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients. Methods We used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy. Results Monotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC). Conclusions These results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non–small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334–43. ©2016 AACR.

High-risk human papillomavirus (HPV)–associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV− SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen–specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP. Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327–39. ©2018 AACR.

Immune checkpoint blockade is effective in “hot” tumors like melanoma with pre-existing immune infiltrates; however, “cold” tumors like prostate cancer fail to respond. We found that prostate cancers harbor regions of hypoxia that resist T cell infiltration even in the context of anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) and anti-PD-1 (programmed cell death protein 1) blockade. These hypoxic zones serve as islands of immune privilege through the recruitment and suppressive polarization of immature myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We found that targeted hypoxia ablation using TH-302, a hypoxia-activated prodrug, sensitized both transplantable and spontaneous models of prostate cancer to checkpoint blockade, coincident with enhanced T cell infiltration and effector function and loss of MDSC recruitment and suppressive function. Tumors treated with the combination of TH-302 and checkpoint blockade showed a reduced capacity to suppressively polarize new myeloid immigrants, implying a durable reconditioning of the tumor microenvironment (TME) into an immune-infiltrated, pro-inflammatory milieu. T cells infiltrating combination-treated tumors exhibited increased mitochondrial respiration, consistent with creation of a metabolically favorable milieu for T cell function. Based on these findings, we hypothesized that other approaches capable of metabolically rewiring the TME should promote anti-tumor immunity and sensitize checkpoint blockade-resistant tumors to immunotherapy. With this in mind, we performed a longitudinal study comparing a panel of different mitochondrial respiration inhibitors and a glutaminase inhibitor for their efficacy in reducing hypoxia, improving T cell infiltration and decreasing myeloid cell recruitment and suppressive polarization using immunofluorescence staining and confocal microscopy. Our preliminary data suggests that inhibitors targeting mitochondrial respiration, rather than those targeting glutamine metabolism synergize with checkpoint blockade and exhibit the highest efficacy in increasing T cell recruitment. We continue to characterize the dynamics of hypoxia reduction, duration of normalization following drug withdrawal, and impact on the immune microenvironment of these diverse approaches to metabolic reconditioning. Citation Format: Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey Ager, Courtney Nicholas, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael Curran. Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5011.