Drug-induced dedifferentiation towards drug-tolerant persister states is a common mechanism cancer cells exploit to escape therapies, hindering durable responses. How early epigenomic and transcriptomic programs coordinate to initiate these reversible transitions remains largely unexplored. Here we employ high-temporal-resolution multi-omics profiling, information-theoretic approaches, and dynamic system modeling to probe these processes in BRAF-mutant melanoma models and patient specimens. We uncover a hysteretic transition trajectory in response to oncogene inhibition and subsequent release, driven by two tightly coupled transcriptional waves that orchestrate genome-scale chromatin reconfiguration. Modeling of these waves suggests NF-κB/RelA-driven chromatin remodeling as the underlying mechanism of cell-state dedifferentiation, which we validate experimentally. We identify RelA-target genes epigenetically modulated to drive this process and define a quantitative epigenome gauge of melanoma cell-state plasticity that supports targeting epigenetic machineries to potentiate oncogene inhibition. Across additional cancer models, oxidative stress-mediated NF-κB/RelA activation emerges as a common driver of transitions into drug-tolerant persister states, revealing a central role for NF-κB axis in coupling oxidative stress to cancer progression.

Quantification of CD3+CD8+ (CD8 T-cells) and (B) CD3+CD4+ (CD4 T-cells) in both tumors and spleens in YUMM2.1; gating strategy after exclusion of dead cells of CD3+CD8+ and CD3+CD4+ cells after gating for CD3+ cells; quantification of CD3+CD8+ (CD8 T-cells) in MC38 and YUMM2.1 spleens; CD3+CD4+ (CD4 T-cells) in MC38 and YUMM 2.1 spleens CD8 T-cells in both tumors and spleens in YUMM1.1.

IGV plot of RNA-Seq from YUMM1.1, YUMM1.7 and YUMM2.1; western blot analysis of cytoplasmic and nuclear beta-catenin in YUMM1.7 and YUMM2.1 cell lines with or without exposure to 10 uM 4HT for 48 hours; top-flash activity of total beta-catenin in YUMM1.7 and YUMM2.1 with or without exposure to 10uM 4HT for 48 hours; representative immunofluorescence of beta-catenin stained non-treated tumors.

PDF file - 32K, Expression of CSF-1 by SM1.

Supplementary figure legends

Gating strategy of CD11c+B220-, CD11c+B220+, CD11c+B220-CD8+ and CD11c+B220-CD103+ cells; gating strategy of CD11b+MHC-IIhigh DCs in CD11c+ cells; gating strategy of CD11b+F4/80+TAMs, CD11b+F4/80+MHC-IIlow TAMs and CD11b+F4/80+MHC-IIhigh TAMs; gating strategy of MO-MDSC (CD11b+Ly6ChighLy6Glow) and PMN-MDSC (CD11b+Ly6ClowLy6Ghigh); gating strategy of Tregs (CD4+CD25+FoxP3+); corresponding normalized enrichment scores (NES), P values and false discovery rates (FDR) of the GSEA plots for YUMM2.1 versus YUMM1.1 enriched pathways involved in immune response, cytokine production and inflammatory response.

PDF file - 65KB, Table S1 Summary for TCR V-beta CDR3 sequence analysis. Table S2 Number of unique Productive Sequences in Top 25% abundance at four different timepoints. Table S3 Number of unique productive Sequences in Top 25% clones at baseline and at Day 30-60.

<p>PDF file - 247KB, MAPK pathway is almost inactive in resting PBMCs.</p>

Abstract A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced in vitro by treatment with MART1 T cell receptor–expressing T cells or with TNFα, and that the phenotype is reversible with withdrawal of inflammatory stimuli. This supports the hypothesis that acquired resistance to cancer immunotherapy can be mediated by inflammation-induced cancer dedifferentiation. Significance: We report a patient whose metastatic melanoma underwent inflammation-induced dedifferentiation as a resistance mechanism to ACT to the MART1 antigen. Our results suggest that future melanoma ACT protocols may benefit from the simultaneous targeting of multiple tumor antigens, modulating the inflammatory response, and inhibition of inflammatory dedifferentiation-inducing signals. Cancer Discov; 8(8); 935–43. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 899

<p>PDF file - 206KB, Restoration of the MAPK pathway activity by vemurafenib in lymphocytes exposed to PD-L1.</p>

PDF file - 68K, Gating strategy for the studies of myeloid cells.

PDF file - 490K, Effects of PLX3397 on the distribution of adoptively transferred lymphocytes in the OT-1 ACT model.

PDF file - 59K, Combined anti-tumor activity of adoptive cell transfer (ACT) immunotherapy and PLX3397 in SM1 and B16 murine melanoma.

PDF file - 48KB, Lack of correlations between changes in ALC (x axis) and changes in the unique productive sequences (y axis) expressed in % of change both. Each dot corresponds to a GA patient.

PDF file - 411KB, Scatter plots for all patients with a rank-rank analysis . For every GA patient a dot plot was generated ranking each single clone according to abundance in baseline (x axis) and post-treatment (y axis). Values range from highest frequency (0) to lowest frequency.

IGV plot exome sequencing from YUMM1.1, YUMM1.7 and YUMM2.1, tumor growth curve of YUMM1.7 and B16 with 4 mice in each group, analysis of the non-synonymous mutational load compared to a strain-matched normal with known dbSNP variants excluded.

Abstract Purpose: To evaluate the immunomodulatory effects of cytotoxic T–lymphocyte-associated protein 4 (CTLA4) blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). Experimental Design: We used next-generation sequencing to study the complementarity-determining region 3 (CDR3) from the rearranged T-cell receptor (TCR) variable beta (V-beta) in PBMCs of 21 patients, at baseline and 30 to 60 days after receiving tremelimumab. Results: After receiving tremelimumab, there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (P = 0.01) and for Shannon index diversity (P = 0.04). In comparison, serially collected PBMCs from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over 1 year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared with patients without toxicity (P = 0.05). No relevant differences were noted between clinical responders and nonresponders. Conclusions: CTLA4 blockade with tremelimumab diversifies the peripheral T-cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system. Clin Cancer Res; 20(9); 2424–32. ©2014 AACR.

Background: The combination of immune checkpoint modulators with chemotherapy improves efficacy compared with chemotherapy alone in PD-L1+ advanced TNBC (IMpassion130; KEYNOTE-355). The addition of IPAT to paclitaxel (PAC) improved efficacy in a phase 2 trial in advanced TNBC (LOTUS). Preliminary overall response rate (ORR) data from a multicenter phase 1b study (NCT03800836) evaluating a triplet combination of IPAT, atezolizumab, and taxane chemotherapy showed promising anti-tumor activity in a similar patient population, irrespective of PD-L1 status [Schmid, AACR 2019]. Here, we report follow-up results including progression-free survival (PFS) from this study. Patients and Methods: Eligible patients had measurable unresectable locally advanced/metastatic TNBC, ECOG performance status 0/1, and had received no prior systemic therapy for advanced disease (prior [neo]adjuvant chemotherapy and/or radiation permitted if all chemotherapy was completed ≥12 months before first dose). Patients with brain metastases were excluded. Patients received oral IPAT 400 mg/day on days 1–21 and IV atezolizumab 840 mg on days 1 & 15 in combination with PAC 80 mg/m2 (Arm A) or nab-PAC 100 mg/m2 (Arm B) on days 1, 8, & 15. Cycles were repeated every 28 days until loss of clinical benefit, unacceptable toxicity, or consent withdrawal. Arms C and D evaluated sequential regimens comprising a doublet induction therapy with the third agent added on day 15 (Arm C: IPAT + PAC, then + atezolizumab; Arm D: atezolizumab + PAC, then + IPAT). Tumors were assessed every 8 weeks. Key endpoints were confirmed ORR (per RECIST v1.1), duration of response (DoR), PFS, and safety. Results: At the data cut-off (26 Jul 2020), results were available from 114 patients (Arm A n=70, Arm B n=20, Arm C n=12, Arm D n=12). Median duration of follow-up was 11.1 months. Efficacy results are summarized in the table. Safety of the combination appeared to be consistent with the known safety profile of the individual drugs. Grade ≥3 adverse events (AEs) occurred in 55% of patients (including rash [13%], diarrhea [12%], and neutropenia [10%]) and serious AEs in 34%. AEs led to discontinuation of IPAT in 6% of patients and atezolizumab in 4%. No new safety signals were identified. Conclusions: Updated results demonstrate a lower ORR than in the preliminary report of the first 26 patients. Subgroup analyses according to PD-L1 or PIK3CA/AKT1/PTEN alteration status or taxane backbone show no consistent trend across endpoints, although small sample sizes limit interpretation. Further biomarker analyses focusing on subgroups and biology may identify subsets of patients deriving a benefit. Citation Format: Peter Schmid, Peter Savas, Enrique Espinosa, Valentina Boni, Antoine Italiano, Shane White, Karen Cheng, Lisa Lam, Lidia Robert, Victor Laliman, Kalpit Shah, Marie-Paule Sablin. Phase 1b study evaluating a triplet combination of ipatasertib (IPAT), atezolizumab, and a taxane as first-line therapy for locally advanced/metastatic triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-28.

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAFV600E mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population.

Abstract Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line Cancers commonly develop resistance against chemotherapeutics or targeted therapies through various types of genetic or non-genetic mechanisms. Non-genetic mechanisms have been shown to occur early on and can provide a latent reservoir of cells for the emergence of various different type of mechanisms, yet very limited understanding of process were resolve main from bulk analysis. Considering the heterogeneous nature of the tumor cells, a single-cell level characterization of the process worth detailed further investigation. Using MAPK inhibition of BRAF-mutant melanomas as a model system, we resolved that cells take different paths to go from drug-sensitive to drug-resistant state. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naïve and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naïve cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies. Citation Format: Yapeng Su, Guideng Li, Melissa Ko, Hanjun Cheng, Ronghui Zhu, Min Xue, Lidia Robert, Raphael Levine, Antoni Ribas, Garry Nolan, Wei Wei, Sylvia Plevritis, David Baltimore, James R. Heath. Systems biology for investigating drug resistance mechanism of melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6585.