This work establishes for the first time that BRAFi promotes myeloid-mediated induction of T cell activation, which is lost at resistance but can be rescued with the addition of CDDO- me, and provides the foundation for its potential use in combination therapies for melanoma.
Abstract
Melanoma is responsible for 80% of skin cancer-related deaths. Approximately 50% of melanoma patients carry the BRAFV600E mutation, which promotes tumor growth. While the use of BRAF inhibitors (BRAFi) has improved outcomes, acquired resistance is a persistent clinical challenge and the impact of BRAFi resistance on the immune tumor microenvironment (iTME) is incompletely understood. We have shown that the addition of the synthetic triterpenoid CDDO-Me to the BRAFi PLX4720 arrested resistance and significantly reduced tumor burden, while CDDO-Me as a single agent or in combination with BRAFi prior to resistance was ineffective. Thus, we hypothesize that this disparity in treatment efficacy may be attributable to temporally regulated BRAFi treatment-induced changes to the iTME.
Drug-induced changes in the iTME pre and post-resistance were characterized in tumors of engrafted transgenic BRAF/Pten mice using scRNA-seq, flow cytometry, and ELISA. T cell proliferation assays were used to assess functional changes in tumor-conditioned myeloid cells.
BRAFi treatment attenuated immunosuppressive myeloid activation pre-resistance, while BRAFi resistance was associated with enhanced tumor-associated macrophage (TAM) recruitment and immunosuppressive myeloid subset activation. However, the addition of CDDO-Me to BRAFi at resistance reversed these outcomes and rescued the ability of TAMs to induce T cell proliferation.
The iTME changes with tumor progression and responds dynamically to BRAFi treatment and resistance. This work establishes for the first time that BRAFi promotes myeloid-mediated induction of T cell activation, which is lost at resistance but can be rescued with the addition of CDDO-Me. Because BRAFi resistance and iTME immunosuppression is reversed by CDDO-Me treatment, these results provide the foundation for its potential use in combination therapies for melanoma.
Dartmouth Health Cancer Center Developmental Funding (Prouty Pilot Grant)
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Advanced BRAF-mutant cutaneous melanoma can be treated with targeted therapy when immune checkpoint inhibitors (ICIs) fail or are not a feasible option. Nevertheless, most patients do not achieve a durable response, highlighting the critical need for therapeutic partners that enhance the long-term efficacy of targeted therapy. Transcriptomic analysis of a BRAF-mutant melanoma model of acquired resistance identified P-selectin glycoprotein ligand-1 (PSGL-1) as a top-upregulated immune mediator upon resistance acquisition. PSGL-1 is a key regulator of CD8+ T cell exhaustion and differentiation, and its inhibition has been shown to enhance T cell function across multiple disease models. Based on these observations, we hypothesized that combined targeting of BRAF/MEK and PSGL-1 would improve anti-tumor responses. Here, we demonstrate that dual inhibition of BRAF/MEK and PSGL-1 elicits durable tumor control in a preclinical model of PD-1-refractory cutaneous melanoma. Single-cell RNA sequencing of the tumor microenvironment reveals robust reprogramming of intratumoral CD8+ T cells toward a less terminally differentiated, memory-like phenotype following combined BRAF/MEK and PSGL-1 targeting. Consistent with these findings, CD8+ T cells in the tumor-draining lymph nodes of PSGL-1-/- mice exhibit enhanced functionality and a less differentiated state of exhaustion when compared with wild-type mice. To extend these observations to a translationally relevant setting, we further show that antibody-mediated blockade of PSGL-1, in combination with BRAF/MEK inhibition, yields superior anti-tumor activity compared with either monotherapy. Collectively, these findings identify PSGL-1 as a promising therapeutic target to enhance the durability of targeted therapy and provide a strong rationale for future clinical evaluation.
O. E. El Naggar, Bn. Ha, ML Rakoto et al.· bioRxiv· 0 citations
This work found that BRAF/MEK inhibitors significantly upregulate TFAP2A, and identifies TFAP2A as a shared driver of both targeted therapy resistance and immunosuppression, offering a one-stone-two-bird strategy to overcome drug resistance and elicit antitumor immunity.
Haiwei Mou, Veronika Yakovishina, Kristen M DeRosa et al.· bioRxiv· 0 citations
Combined BRAF/MEK and PSGL-1 targeting provides a durable anti-tumor response through enhanced functionality and a memory-like phenotype in CD8⁺ T cell subsets.
Mutations in the BRAF (B-raf serine-threonine-protein kinase) gene occur in the majority of melanoma patients and significantly contribute to the progression of the disease. One of the most commonly used methods for treating melanoma is the use of BRAF and MEK (mitogen-activated protein kinase kinase) inhibitors. Unfortunately, resistance to treatment quickly develops in most patients. Therefore, an exhaustive understanding of the molecular basis of this resistance is crucial. We developed two melanoma cell lines resistant to vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor), which we previously characterized as highly invasive. Because cancer cells acquiring resistance may undergo significant metabolic changes, we investigated these processes in the obtained melanoma double-resistant cells. These cells exhibit increased glycolysis and elevated caveolin 1 levels, which contribute to their increased invasiveness. The mitochondria present in the peripheral region of the resistant cells are more functional than those present in the same region of control cells. Resistant cells also form more lipid droplets, but they are smaller than in control cells. Cholesterol ester level is reduced in resistant cells, while free cholesterol level is elevated. Moreover, resistant cells exhibit reduced lipolysis rate and expression of proteins regulating this process. In summary, the obtained results suggest that the purpose of metabolic changes present in melanoma cells resistant to BRAF/MEK inhibitors is mainly to support the significantly increased invasiveness of these cells.
M. Kot, A. Simiczyjew, Katarzyna Pietraszek-Gremplewicz et al.· Biochimica et biophysica act...· 0 citations
Despite advancements in immunotherapy, more than half of melanoma patients will either not respond or will later relapse after treatment with immune checkpoint blockade (ICB). For later-stage patients, chemotherapy is an essential treatment modality, but tumor intrinsic cell survival mechanisms and an immunosuppressive tumor microenvironment often limit efficacy. We recently discovered that by upregulating the secretion of Pros1, tumor cells limit the response of macrophages to chemotherapy released tumor Damage Associated Molecular Patterns (DAMPs). By pharmacologically inhibiting Ptp1b, a signaling intermediary downstream of the Mer receptor, macrophage responsiveness to DAMPs can be restored. This is associated with increased macrophage M1 polarization and immune infiltration, as well as a 40-80% decrease in tumor volume in multiple preclinical models.
We hypothesize that because Ptp1b inhibition prevents tumor suppression of DAMP responsiveness, combining Ptp1b inhibition may improve the efficacy of multiple chemotherapies, particularly those that promote necrotic and immunogenic cell death. To test this, macrophages were co-cultured with murine melanoma cells (B16F10) and cisplatin, doxorubicin, or dacarbazine in the presence or absence of Ptp1b inhibitor via transwell assay, after determining the relative amounts of apoptosis/necrosis produced by each chemotherapy.
Preliminary results indicate that Ptp1b inhibition significantly increases macrophage pro-inflammatory gene expression by 7.5-fold in a setting of chemotherapy-induced tumor DAMP release. This indicates that inhibiting Ptp1b could synergistically improve chemotherapy effectiveness.
As Ptp1b inhibitors have a reasonable safety profile based on multiple clinical trials, combining Ptp1b inhibiting drugs with chemotherapy may be a novel way to restore the innate immune response during treatment while also improving patient outcomes.
NCI- RO1 CA262241(Ubil)
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Oluwaseyi Omodiminiyi, Nestor Prieto-Dominguez, Eric Ubil· Journal of Immunology· 0 citations
Background The clinical significance of clonal hematopoiesis of indeterminate potential (CHIP) in melanoma remains incompletely defined, particularly with respect to CHIP genotype, clone size, and somatic mutations (e.g BRAF mutations). We integrated human cohort data and a syngeneic melanoma mouse model to evaluate whether CHIP is associated with melanoma risk, tumor growth, and differential clinical outcomes. Methods We analyzed CHIP prevalence and survival in a large treatment-unselected melanoma cohort (n=2,480), evaluated tumor growth in a syngeneic BRAF-mutant (BRAFmut) melanoma murine model of TET2-CHIP and DNMT3A-CHIP, and assessed survival outcomes in an immune checkpoint inhibitor (ICI)-treated advanced melanoma cohort (n=361). Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Kaplan-Meier analyses and multivariable Cox proportional hazards models. Results CHIP was enriched among patients with treatment-unselected melanoma compared with age/sex-matched healthy controls, and larger CHIP clone size showed an age-adjusted association with inferior OS. In a syngeneic BRAFmut melanoma murine model, TET2-CHIP, but not DNMT3A-CHIP, was associated with significantly increased primary melanoma tumor growth. Among patients with ICI-treated advanced melanoma, CHIP was associated with worse OS compared with patients without CHIP. TET2-CHIP had the strongest adverse association with survival, whereas DNMT3A-CHIP was not significantly associated with PFS or OS. Conclusions CHIP is enriched in melanoma and exploratory analyses demonstrate genotype-specific differences in melanoma tumor growth and clinical outcomes. These findings support further investigation of genotype-specific CHIP profiling as a potential biomarker for melanoma risk stratification and immunotherapy outcomes.
M. Alford-Holloway, S. Reed, Y. Pershad et al.· medRxiv· 0 citations
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