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Purpose: Eganelisib (IPI-549) is a first-in-class, orally administered, highly selective PI3Kγ inhibitor with antitumor activity alone and in combination with programmed cell death protein 1/ligand 1 (PD-1/PD-L1) inhibitors in preclinical studies. This phase 1/1b first-in-human, MAcrophage Reprogramming in Immuno-Oncology-1 (NCT02637531) study evaluated the safety and tolerability of once-daily eganelisib as monotherapy and in combination with nivolumab in patients with solid tumors. Patients and Methods: Dose-escalation cohorts received eganelisib 10–60 mg as monotherapy (n = 39) and 20–40 mg when combined with nivolumab (n = 180). Primary endpoints included incidence of dose-limiting toxicities (DLT) and adverse events (AE). Results: The most common treatment-related grade ≥3 toxicities with monotherapy were increased alanine aminotransferase (ALT; 18%), aspartate aminotransferase (AST; 18%), and alkaline phosphatase (5%). No DLTs occurred in the first 28 days; however, toxicities meeting DLT criteria (mostly grade 3 reversible hepatic enzyme elevations) occurred with eganelisib 60 mg in later treatment cycles. In combination, the most common treatment-related grade ≥3 toxicities were increased AST (13%) and increased ALT and rash (10%). Treatment-related serious AEs occurred in 5% of monotherapy patients (grade 4 bilirubin and hepatic enzyme increases in one patient each) and 13% in combination (pyrexia, rash, cytokine release syndrome, and infusion-related reaction in ≥2 patients each). Antitumor activity was observed in combination, including patients who had progressed on PD-1/PD-L1 inhibitors. Conclusions: On the basis of the observed safety profile, eganelisib doses of 30 and 40 mg once daily in combination with PD-1/PD-L1 inhibitors were chosen for phase 2 study.

PURPOSE Combination programmed cell death protein 1/cytotoxic T-cell lymphocyte-4–blockade and dual BRAF/MEK inhibition have each shown significant clinical benefit in patients with BRAFV600-mutant metastatic melanoma, leading to broad regulatory approval. Little prospective data exist to guide the choice of either initial therapy or treatment sequence in this population. This study was conducted to determine which initial treatment or treatment sequence produced the best efficacy. PATIENTS AND METHODS In a phase III trial, patients with treatment-naive BRAFV600-mutant metastatic melanoma were randomly assigned to receive either combination nivolumab/ipilimumab (arm A) or dabrafenib/trametinib (arm B) in step 1, and at disease progression were enrolled in step 2 to receive the alternate therapy, dabrafenib/trametinib (arm C) or nivolumab/ipilimumab (arm D). The primary end point was 2-year overall survival (OS). Secondary end points were 3-year OS, objective response rate, response duration, progression-free survival, crossover feasibility, and safety. RESULTS A total of 265 patients were enrolled, with 73 going onto step 2 (27 in arm C and 46 in arm D). The study was stopped early by the independent Data Safety Monitoring Committee because of a clinically significant end point being achieved. The 2-year OS for those starting on arm A was 71.8% (95% CI, 62.5 to 79.1) and arm B 51.5% (95% CI, 41.7 to 60.4; log-rank P = .010). Step 1 progression-free survival favored arm A ( P = .054). Objective response rates were arm A: 46.0%; arm B: 43.0%; arm C: 47.8%; and arm D: 29.6%. Median duration of response was not reached for arm A and 12.7 months for arm B ( P < .001). Crossover occurred in 52% of patients with documented disease progression. Grade ≥ 3 toxicities occurred with similar frequency between arms, and regimen toxicity profiles were as anticipated. CONCLUSION Combination nivolumab/ipilimumab followed by BRAF and MEK inhibitor therapy, if necessary, should be the preferred treatment sequence for a large majority of patients.

Study IPI-549-01 progression-free survival censoring rules for efficacy analyses

Purpose:Eganelisib (IPI-549) is a first-in-class, orally administered, highly selective PI3Kγ inhibitor with antitumor activity alone and in combination with programmed cell death protein 1/ligand 1 (PD-1/PD-L1) inhibitors in preclinical studies. This phase 1/1b first-in-human, MAcrophage Reprogramming in Immuno-Oncology-1 (NCT02637531) study evaluated the safety and tolerability of once-daily eganelisib as monotherapy and in combination with nivolumab in patients with solid tumors.Patients and Methods:Dose-escalation cohorts received eganelisib 10–60 mg as monotherapy (n = 39) and 20–40 mg when combined with nivolumab (n = 180). Primary endpoints included incidence of dose-limiting toxicities (DLT) and adverse events (AE).Results:The most common treatment-related grade ≥3 toxicities with monotherapy were increased alanine aminotransferase (ALT; 18%), aspartate aminotransferase (AST; 18%), and alkaline phosphatase (5%). No DLTs occurred in the first 28 days; however, toxicities meeting DLT criteria (mostly grade 3 reversible hepatic enzyme elevations) occurred with eganelisib 60 mg in later treatment cycles. In combination, the most common treatment-related grade ≥3 toxicities were increased AST (13%) and increased ALT and rash (10%). Treatment-related serious AEs occurred in 5% of monotherapy patients (grade 4 bilirubin and hepatic enzyme increases in one patient each) and 13% in combination (pyrexia, rash, cytokine release syndrome, and infusion-related reaction in ≥2 patients each). Antitumor activity was observed in combination, including patients who had progressed on PD-1/PD-L1 inhibitors.Conclusions:On the basis of the observed safety profile, eganelisib doses of 30 and 40 mg once daily in combination with PD-1/PD-L1 inhibitors were chosen for phase 2 study.

Study IPI-549-01 full protocol inclusion and exclusion criteria

Representativeness of Study Participants

Study IPI-549-01 methodology and criteria for assessing MDSCs

Supplementary Data from Adjuvant Pembrolizumab versus IFNα2b or Ipilimumab in Resected High-Risk Melanoma

Patients with advanced melanoma that is resistant to PD-1 blockade therapy have limited treatment options. Vidutolimod (formerly CMP-001), a virus-like particle containing a CpG-A Toll-like receptor 9 (TLR9) agonist, may reverse PD-1 blockade resistance by triggering a strong IFN response to induce and attract antitumor T cells. In the dose-escalation part of this phase Ib study, vidutolimod was administered intratumorally at escalating doses with intravenous pembrolizumab to 44 patients with advanced melanoma who had progressive disease or stable disease on prior anti–PD-1 therapy. The combination of vidutolimod and pembrolizumab had a manageable safety profile, and durable responses were observed in 25% of patients, with tumor regression in both injected and noninjected lesions, including visceral lesions. Patients who responded to vidutolimod and pembrolizumab had noninflamed tumors at baseline and induction of an IFNγ gene signature following treatment, as well as increased systemic expression of the IFN-inducible chemokine CXCL10.Significance:In this phase Ib study in patients with advanced melanoma, intratumoral TLR9 agonist vidutolimod in combination with pembrolizumab had a manageable safety profile and showed promising clinical activity, supporting the further clinical development of vidutolimod to overcome PD-1 blockade resistance through induction of an IFN response.See related commentary by Sullivan, p. 2960.This article is highlighted in the In This Issue feature, p. 2945

PDF file - 144K, Long-term effects of P124SMEK1 expression on population doubling and p-ERK levels. (A) Impact of regulated expression of P124SMEK1 on long-term growth of M238. Stable lines expressing empty vector, FLAG-MEK1 WT or FLAG-MEK1 P124S were cultured with different levels of doxycycline, enabling low (0.1 ng/ml) or high (0 ng/ml) expression levels of exogenous MEK1. Equal number of cells were serially plated every 4 days and fold growth calculated by dividing the number of viable cells (trypan-blue negative) at the end of each four day period over the starting seeding number. (B) Long-term, regulated expression of WTMEK or P124SMEK and corresponding levels of ERK activation in a clonogenic assay (Fig. 2G). Doxycycline-controlled expression of vector (control), FLAG-MEK1 WT, vs. FLAG-MEK1 P124S in the V600EB-RAF melanoma cell line, M238. Protein lysates (192 h post seeding at 0, 0.1, and 100 ng/ml doxycycline) were probed by Western blotting for the indicated phospho- and total protein levels. Tubulin, loading control.

XLSX file 16K, WES data characteristics including library construction, total reads, base coverage, capture specificity

PDF file - 76K, Relative expression levels of WT vs. P124SMEK1 in the YUKSI B-RAF/MEK1 double-mutant melanoma short-term culture. Genomic DNA (gDNA) and cDNA derived from YUKSI were subjected to exon 3 sequencing by the Sanger method. Relative peaks of WT vs. P124SMEK1 is shown to be 1:1 for both gDNA and cDNA based on nucleotide chromatograms, showing no allelic bias in RNA expression.

PDF file - 1292K, Study scheme, assay validation, functional changes of patients F5-2 and F5-8.

Quantitative CD8+ Counts in patients with Liver and skin metastases.

PDF file 6308K, This includes all supplementary figures except S2 and contains additional clinical data, sequence and copy number analysis as well as immunohistochemical and functional studies

PDF file - 46K, Rapid symptomatic relief in patient 12 treated with GSK2118436/dabrafenib. This patient's right axillary nodal tumor was punch-biopsied at baseline (prior to B-RAFi therapy), showing concurrent B-RAF/MEK1 mutations, and became non-palpable after eleven days of continuous GSK2118436 dosing. This tumor shrinkage was associated with increased right shoulder mobility, decreased pain and right axillary and breast swelling, despite the best overall response in this patient (Table 1) not achieving partial response by RECIST criteria.

Tumor responses to programmed cell death protein 1 (PD-1) blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, an antibody to PD-1. Biopsies were dissociated, and single-cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single-cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells significantly increased in patients' biopsies taken on treatment. The percentage of cells with a regulatory T-cell phenotype, monocytes, and natural killer cells did not change while on PD-1 blockade therapy. CD8+ memory T cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4+ effector memory T cells significantly decreased on treatment, whereas CD4+ effector T cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 was detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and myeloid-derived suppressor cells in tumors, with the CD8+ effector memory T-cell subset being the major T-cell phenotype expanded in patients with a response to therapy. Cancer Immunol Res; 4(3); 194–203. ©2016 AACR.

Univariate Analysis of Pretreatment prognostic factors in the Discovery cohort.

Supplementary Figure 1. Exome sequencing and copy number changes in M431 and corresponding whole-tumor biopsy. Supplementary Figure 2. Mutations in antigen presentation machinery from anti-PD1 treated melanoma cohort. Supplementary Figure 3. Immunohistochemistry (IHC) analysis of biopsies from melanoma metastases with a JAK1 loss of function mutation. Supplementary Figure 4. Selection of PD-L1 flow cytometry antibody and impact of phosphatase inhibitor as well as temperature on measuring surface PD-L1 expression and pAKT. Supplementary Figure 5. Flow cytometry gating strategy and dose response curve of interferon alpha, beta and gamma to determine the optimal concentrations. Supplementary Figure 6. Time course of PD-L1 surface expression upon interferon alpha, beta and gamma treatment for selected cell lines to determine the optimal time point for the screening. Supplementary Figure 7. Time course of PD-L1 expression upon interferon alpha, beta or gamma treatment for the cell lines with poor or no up-regulation upon 18 hours exposure. Supplementary Figure 8. Interferon signaling pathway in good and poorly responding cell lines. Supplementary Figure 9. PD-L1 expression upon interferon alpha and beta exposure. Supplementary Figure 10. Predicted functional consequences of M368 JAK2 D313 splice site mutation. Supplementary Figure 11. Immunohistochemistry (IHC) analysis of biopsies from melanoma metastases with a JAK1 loss of function mutation. Supplementary Figure 12. JAK1 wild-type lentiviral vector transduction. Supplementary Figure 13. Mutations in antigen presentation machinery from anti-PD1 treated colorectal cohort. Supplementary Figure 14. DNA damage repair gene mutations in endometrial cancer cell lines with JAK1/2 mutations. Supplementary Figure 15. Frequency of JAK1 and JAK2 alterations and their association with overall survival in additional TCGA datasets. Supplementary Figure 16. Functional effect of genetic loss of reactive PD-L1 on responses to PD-1 blockade. Supplementary Database 1. Whole exome sequencing of 23 baseline biopsies from patients analyzed in Fig. 1, and whole exome sequencing of the M431 cell line (Accession code: SRP067938).

PDF file - 188K, Effects of MEK1 P124S co-expression with B-RAF V600K on cellular p-ERK levels and growth inhibitory sensitivities to B-RAFi and MEKi. (A) The YULAC V600KB-RAF melanoma cell line was stably introduced with the doxycycline-repressible tetracycline transactivator (tTA) and subsequently with vector, FLAG-MEK1, or FLAG-MEK1 P124S expression constructs. Stable cell lines incubated with doxycycline (100 ng/ml) or washed from doxycycline for 2 days were lysed, and the lysates probed for levels of indicated proteins by Western blotting. Tubulin, loading controls. (B) Survival curves for indicated YULAC stable cell lines treated with the indicated PLX4032 or AZD6244 for 72 h in the presence of doxycycline (top) or its absence (bottom). Data represent percent surviving cells relative to DMSO-treated controls (mean +/- SEM, n = 5). The dashed line corresponds to 50% cell killing.

PDF file - 181K, Patient tissues harboring concurrent and somatic B-RAF and MEK1 mutations. Chromatograms showing B-RAF exon 15 at V600 codon and MEK1 exon 3 at I111 or P124 codons in patient-matched normal tissue, baseline melanomas (prior to B-RAFi therapy), and melanomas on disease progression (i.e. at acquisition of B-RAFi resistance). Specific codons in red; N/A, not available.

Supplementary Figure Legends

PDF file - 76K, B-RAFi and MEKi combinatorial treatments lead to strong synergistic growth inhibition of V600EB-RAF cell lines regardless of concurrent P124SMEK1 expression. M229 and M238 vector vs. MEK1 stable cell lines were cultured with different levels of doxycycline (ng/ml), treated with constant ratios of B-RAFi (vemurafenib) and MEK1/2i (selumetinib), as indicated (muM), and measured for survival after 72h. Relative synergy of indicated drug combinations (expressed as log10 of CI values) is shown. Calculations were made by CalcuSyn software using the method of Chou and Taladay. Interpretation of CI values is summarized as follows: CI < 0.1 (very strong synergy); = 0.1-0.3 (strong synergy); = 0.3-0.7 (synergy); 0.7-0.85 (moderate synergy); = 0.85-0.9 (slight synergy); = 0.90-1.10 (nearly additive); and = 1.10-1.20 (slight antagonism). The relevant correlated Log10 (CI) values are shown as follow: Log10 (CI 0.1) = -1; Log10 (CI 0.3) = -0.5228787452803376; Log10 (CI 0.7) = -0.1549019599857432, and Log10 (CI 0.85) = -0.07058107428570727.

PDF file - 131K, Protein and cell specificity studied, comparison of polyfunctional T cells and other cells.

Gating strategy to study T cells subpopulations within tumor-infiltrating lymphocytes and PBMC. We represent dot-plots on the top of the figure, and a dendrogram displaying the gating strategy of eight phenotype markers, before applying Boolean gates, and posteriorly analyzed with the exhaustive expansion software. During gating by morphology, and excluding duplex, double positive live and CD3+ T cells were gated on CD8, CD4, CD45RA, CD45RO, CCR7, CD62L, CD27, CD57, CD95 and PD-1.

PDF file 8842K, Supplementary Figure S2 contains Sanger re-sequencing to estimate the specificity of WES

Fluorochromes used on this study

PDF file - 181K, WTMEK and P124SMEK activated by V600EB-RAF display similar in vitro kinase activities toward recombinant ERK1. HEK293T cells were transfected with vector, FLAG-MEK1 WT, or FLAG-MEK1 P124S in the absence or presence of co-transfected V600EB-RAF. Cells were lysed and exogenous MEK1 immunoprecipitated (IP) using isotype (I) control or anti-FLAG (F) antibody (ab), washed, and subjected to in vitro kinase assay. Pre-IP lysates were immunoblotted for FLAG levels. After the kinase reaction, the supernatant (sup) or ab-bead fractions were mixed with denaturing loading buffers, fractionated and immunoblotted against indicated phospho- or total protein levels (rERK, recombinant ERK).