Protein kinase Cα-mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it.

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.

Histiocyte predominant myocarditis resulting from the addition of interferon gamma to cyclophosphamide-based lymphodepletion for adoptive cellular therapy.

BACKGROUND: Adoptive cellular therapy (ACT) is a promising treatment for synovial sarcoma (SS) with reported response rates of over 50%. However, more work is needed to obtain deeper and more durable responses. SS has a 'cold' tumor immune microenvironment with low levels of major histocompatibility complex (MHC) expression and few T-cell infiltrates, which could represent a barrier toward successful treatment with ACT. We previously demonstrated that both MHC expression and T-cell infiltration can be increased using systemic interferon gamma (IFN-γ), which could improve the efficacy of ACT for SS. CASE PRESENTATION: We launched a phase I trial incorporating four weekly doses of IFN-γ in an ACT regimen of high-dose cyclophosphamide (HD Cy), NY-ESO-1-specific T cells, and postinfusion low-dose interleukin (IL)-2. Two patients were treated. While one patient had significant tumor regression and resultant clinical benefit, the other patient suffered a fatal histiocytic myocarditis. Therefore, this cohort was terminated for safety concerns. CONCLUSION: We describe a new and serious toxicity of immunotherapy from IFN-γ combined with HD Cy-based lymphodepletion and low-dose IL-2. While IFN-γ should not be used concurrently with HD Cy or with low dose IL-2, IFN-γ may still be important in sensitizing SS for ACT. Future studies should avoid using IFN-γ during the immediate period before/after cell infusion. TRIAL REGISTRATION NUMBERS: NCT04177021, NCT01957709, and NCT03063632.

Systemic Interferon-γ Increases MHC Class I Expression and T-cell Infiltration in Cold Tumors: Results of a Phase 0 Clinical Trial.

Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer-testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into "hot" tumors will work in concert with anti-PD-1 therapy to provide patient benefit.