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Fig. S1. Establishment of SB-3123 murine melanoma model. Fig. S2. Schematic of primer design for BRAF c.1799T > A (V600E) mutation analysis in SB-3123p murine melanoma cells. Fig. S3. SB-3123p melanoma is highly sensitive to vemurafenib. Fig. S4. BRAFV600E specific effect of vemurafenib upon SB-3123p established melanomas. Fig. S5. Establishment of SB-3123 murine melanoma cells expressing mouse gp100. Fig. S6. Concurrent vemurafenib therapy is required for sustained tumor growth control. Fig. S7. SB-3123 proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines. Fig. S8. Isobologram analysis from our 8x8 screen for the synergistic (black circle) drug combination of vemurafenib + IFN-γ/TNF-α. Fig. S9. IFN-γ and TNF-α combined with vemurafenib induce growth arrest of SB-3123 melanoma cells. Fig. S10. Melanoma proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines in a BRAFV600E dependent manner. Fig. S11. Human melanoma proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines in a BRAFV600E dependent manner. Fig. S12. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature enriched in genes induced by IFN-γ/TNF-α treatment of SB3123. Supplementary Table 1 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 1. Supplementary Table 2 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 2. Supplementary Table 3 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 3. Supplementary Table 4 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 4. Supplementary Table 5 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 5. Table S6. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature normalized to the vemurafenib only dependent gene expression signature in SB3123. Table S7. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature normalized to the IFN- γ/TNF-α only dependent gene expression signature in SB3123. Table S8. Gene set enrichment analysis of vemurafenib dependent gene expression signature normalized to control treated SB3123. Supplementary Table 9 - IFNy/TNFB induced genes vs Control Treatment, fold change > 2.0, p-value < 0.01. Supplementary Table 10 - Vemurafenib induced genes vs control treatment, fold change > 2.0, p-value < 0.01. Supplementary Table 11 - Vem + IFNg/TNFa vs IFNg/TNFa differentially expressed genes, FC > 1.5.

Supplementary Materials and Methods

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Purpose: Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γc) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified.Experimental Design: To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (TCM), effector memory (TEM), and stem cell memory (TSCM) subsets on the strength of tumor regression as well as the dose and type of clinically available γc cytokines, including IL-2, IL-7, IL-15, and IL-21.Results: We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γc cytokine only moderately correlated with response.Conclusion: Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γc cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo. Clin Cancer Res; 17(16); 5343–52. ©2011 AACR.

Supplemental Figure S1. Clinical strategy to pharmacologically inhibit Akt during ex vivo expansion of tumor-infiltrating lymphocytes (TIL).

Supplementary Figure 5 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplemental Figure S4. Akti-treated tumor-specific Pmel CD8+ T cell have reduced phosphorylation of Akt and substrates in presence of pharmacologic inhibition of Akt.

Supplementary Figure 4 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplemental Figure S2. CD8+ tumor-infiltrating lymhphocytes (TIL) isolated from patient with melanoma have reduced phosphorylation of Akt T308 and GSK3βS9 in presence of Akt inhibitor.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer. Cancer Res; 75(2); 296–305. ©2014 AACR.

Supplementary Figure 2 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplemental Figure S6. 8.Akt inhibition of murine CD8+ T cells increased expression of CD62L and CD27, and decreased expression of CD44.

Supplementary Figure 7 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 3 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplemental Figure S7. Pharmacologic inhibition of Akt modulates global metabolic profile and key lipid metabolites of antigen-experienced murine CD8+ T cells.

Supplemental Figure S3. Surface phenotype of Akti-treated and vehicle TIL isolated from patient with melanoma and expanded to clinical scale for 30 days prior to adoptive-transfer into humanized mouse model.

Supplementary Figure 6 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 1 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Background: Leukemia inhibitory factor (LIF) is an immunosuppressive cytokine linked to tumor growth and metastasis. LIF overexpression correlates with poor prognosis and chemoresistance in multiple tumor types including PDAC. Preclinical data show that LIF promotes an immunosuppressive tumor microenvironment, hindering cytotoxic CD8+ T cell recruitment; low T cell infiltration also correlates with mortality in patients with PDAC. Preclinical studies show LIF-blocking antibodies sensitize tumors to PD-1/PD-L1 inhibition, inhibit epithelial-mesenchymal transition, and prolong survival in combination with CT. In patients with PDAC, higher LIF levels correlate with more aggressive pathology, elevated CA19-9 (a PDAC biomarker), and lower response and survival rates. AZD0171 (formerly MSC-1), a first-in-class, humanized, IgG1 monoclonal antibody, binds specifically and potently to LIF, preventing downstream signaling. In a phase 1 dose escalation study (NCT03490669), AZD0171 monotherapy had manageable safety and led to stable disease (SD) in 34.2% of patients with advanced solid tumors across all dose levels. AZD0171 + CT may improve survival outcomes vs CT alone. Based on preclinical data, AZD0171 may function to stimulate antitumor immune response, and combination with the PD-L1 inhibitor durvalumab could prolong that response and overcome peripheral tolerance in patients with metastatic PDAC. Methods: This is a phase 2, open-label, single-arm, multicenter study of AZD0171 + durvalumab and CT in treatment-naive patients with metastatic PDAC (NCT04999969). Eligible patients must have an ECOG performance status 0 or 1, a Gustave Roussy Immune Score 0 or 1, ≥1 measurable target lesion per RECIST v1.1, and confirmed presence of tumoral CD8+ T cells. Patients with central nervous system metastasis, history of leptomeningeal disease or cord compression, a thromboembolic event ≤3 months prior to study treatment, unresolved grade ≥2 toxicities from prior therapy, or a sensitizing mutation or tumor characteristic for which there is a preferred treatment, are excluded. As PDAC is poorly immune infiltrated, a novel clinical trial assay will be used to select for patients who have existing resident CD8+ T cells and may therefore be more likely to respond. About 115 patients will receive intravenous AZD0171, durvalumab and CT until disease progression or unacceptable toxicity. The primary endpoints are safety and overall survival (OS) rate at 12 months. Secondary endpoints include objective response rate, disease control rate (confirmed response or SD ≥16 weeks), duration of response, median progression-free survival (PFS), PFS rate at 4 months, median OS, pharmacokinetics, pharmacodynamics (changes in serum CA19-9 level and tumor CD8+ T cell infiltration) and immunogenicity. The trial is currently recruiting. Citation Format: Grainne O'Kane, Teresa Macarulla, Fiyinfolu Balogun, Antoine Hollebecque, Matthew J. Reilley, Sreenivasa R. Chandana, Jim Eyles, Oluwaseun Ojo, Philip Overend, Douglas C. Palmer, Nadia Luheshi, Mayukh Das, Antoine Italiano, Joan Seoane. A phase 2 trial of first-line AZD0171 + durvalumab and chemotherapy (CT) in patients with metastatic pancreatic ductal adenocarcinoma (PDAC) and CD8+ T cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT126.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer. Cancer Res; 75(2); 296–305. ©2014 AACR.

Purpose: Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γc) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified. Experimental Design: To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (TCM), effector memory (TEM), and stem cell memory (TSCM) subsets on the strength of tumor regression as well as the dose and type of clinically available γc cytokines, including IL-2, IL-7, IL-15, and IL-21. Results: We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γc cytokine only moderately correlated with response. Conclusion: Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γc cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo. Clin Cancer Res; 17(16); 5343–52. ©2011 AACR.

T-cell–based immunotherapies can be effective in the treatment of large vascularized tumors, but they rely on adoptive transfer of substantial numbers (∼20 million) of tumor-specific T cells administered together with vaccination and high-dose interleukin (IL)-2. In this study, we report that ∼10,000 T cells gene-engineered to express a single-chain IL-12 molecule can be therapeutically effective against established tumors in the absence of exogenous IL-2 and vaccine. Although IL-12–engineered cells did not perist long-term in hosts, they exhibited enhanced functionality and were detected in higher numbers intratumorally along with increased numbers of endogenous natural killer and CD8+ T cells just before regression. Importantly, transferred T cells isolated from tumors stably overproduced supraphysiologic amounts of IL-12, and the therapeutic effect of IL-12 produced within the tumor microenvironment could not be mimicked with high doses of exogenously provided IL-12. Furthermore, antitumor effects could be recapitulated by engineering wild-type open-repertoire splenocytes to express both the single-chain IL-12 and a recombinant tumor-specific T-cell receptor (TCR), but only when individual cells expressed both the TCR and IL-12, indicating that arrested migration of T cells at the tumor site was required for their activities. Successful tumor eradication was dependent on a lymphodepleting preconditioning regimen that reduced the number of intratumoral CD4+ Foxp3+ T regulatory cells. Our findings reveal an approach to genetically modify T cells to reduce the cell number needed, eliminate the need for vaccines or systemic IL-2, and improve immunotherapy efficacy based on adoptive transfer of gene-engineered T cells. Cancer Res; 70(17); 6725–34. ©2010 AACR.

Tumors progress in immunocompetent hosts despite the local infiltration of tumor-specific effector T cells. Recent advances have identified ion transport as important for T cell activation and function. We report that the concentration of potassium is markedly elevated within murine and human tumors in comparison to other tissues while other ions are minimally perturbed. Additionally, ex vivo stimulation of effector T cells in hyperkalemic conditions led to profound suppression of T cell activation, cytokine production, and glycolytic metabolism without affecting viability - suggesting a role for potassium in tumor immune evasion. Characterization of this phenomenon with whole-transcriptome RNA-Sequencing revealed that elevated potassium specifically repressed TCR induced transcripts with the effector cytokine interferon gamma (IFNγ) being among the most depressed. Moreover, hyperkalemia limited TCR induced glucose uptake and consumption, metabolic changes required for full effector function. Prior investigations into the role of potassium transport in cellular physiology have centered on the presumption that the ion's sole function is to maintain cellular membrane potential and calcium influx. However, we find that elevated potassium had no effect on TCR induced calcium signaling. Although hyperkalemia increased the cytoplasmic membrane potential of effector T cells as expected, other methods to similarly depolarize the cell without the addition of extracellular potassium did not produce the inhibitory phenomenon. Therefore, we conclude that the immunosuppressive effect induced by hyperkalemia is independent of potassium's previously understood role in the regulation of cytoplasmic membrane potential and calcium signaling. Causatively, elevated potassium led to blunted serine/threonine phosphorylation within the Akt/mTOR signaling cascade following TCR ligation, while pharmacologic or genetic means to restore Akt signaling rescued effector function. Mechanistically, the suppressive effect of elevated extracellular potassium was directly related to an increase in intracellular levels, and depletion of intracellular potassium restored full T cell activation and cytokine production. T cells genetically reprogrammed to express the potassium efflux channel Kcna3 were resistant to potassium mediated suppression - displaying lower intracellular potassium levels and increased cytokine production. Finally, tumor-specific Kcna3 expressing cells exhibited augmented in vivo intratumoral IFNγ production, Akt/mTOR signaling, and enhanced anti-tumor activity against established melanoma in tumor bearing mice. These results uncover a novel axis of tumor-induced immune suppression via an ‘ionic’ checkpoint. The manipulation of cellular ion transport represents an entirely new approach for cancer immunotherapy. Citation Format: Robert L. Eil, Rahul Roychoudhuri, Madhu Sukumar, David Clever, Jenny H. Pan, Shashank Patel, Douglas C. Palmer, Nicholas P. Restifo. Ionic immune suppression within the tumor microenvironment limits T cell effector function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1463.

DNA damage response and repair (DDR) pathways are central to biological processes like hematopoietic stem cell maintenance, lymphocyte development and adaptive response to inflammation and cancer. However, the role of DDR in antigen experienced T-lymphocytes remains unclear. We studied the dynamics of DDR response during CD8+ T cell proliferation and differentiation. We found that DDR gene transcripts and gamma-H2AX, a marker for DDR pathway activation, are significantly up-regulated in CD8+ T cells expanded in-vitro and in-vivo. Next we aimed to determine the consequence of the activation of DDR pathways. Based on flow cytometry measurements of gamma-H2AX phosphorylation levels, we categorized CD8+ T cells into gamma-H2AXLo and gamma-H2AXHi cells. gamma-H2AXLo CD8+ T cells actively proliferate and produce cytokines, while gamma-H2AXHi CD8+ T cells lose their ability to produce cytokines, and undergo apoptosis. To define whether oxidative stress or replicative stress incurred in CD8+ T cell triggers this activation of DDR, we acutely treated cells with chemical agents and studied functional differences. These studies revealed that oxidative stress may be responsible for the inhibition of cytokine production and increase in cell death. Using Pmel transgenic p53-/- mouse model, we show that by deleting p53, a major signal modulator of DDR, the loss of cytokine production and cell death due to oxidative stress is reverted in CD8+ T cells. Currently, we are exploring the chemical and gene modification methods to manage oxidative stress in CD8+ T cells to improve T-cell based adoptive cell transfer therapies for cancer. Citation Format: Shashank Patel, Madhusudhanan Sukumar, Douglas Palmer, Rahul Roychoudhari, Rabindra Roy, Andre Nussenzweig, Nicholas Restifo. Genomic stress in antigen experienced T-lymphocytes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2015-1347