CDK7 enables adaptive gene transcription required by stromal cells to support distinct T-cell lymphoma phenotypes and represents a novel microenvironment-directed therapeutic target

Abstract Over 75% of PTCL-NOS patients fail to achieve long-term control with current therapies, highlighting the need for novel therapeutic targets. Genetic and non-genetic drivers in cancer cells require external cues from other cancer cells and from the lymphoma microenvironment (LME), which enable cancer cell adaptation to metabolic stress, immune mediated stress and treatment exposure. The resulting signaling crosstalk propels the co-evolution of cancer and LME cells with emergence of diverse cancer-supporting cell states. Identifying these and their maintenance mechanisms may therefore reveal novel therapeutic targets. We characterized the LME of PTCL using transcriptional signature deconvolution and multiparametric imaging in 845 patient samples. We identified four biologically and clinically distinct categories named Depleted (DP), B-cell rich (BR), Mesenchymal (MS) and Inflammatory (IN) based on the proportion of different LME cells. RNA-seq analysis showed that SMA+ cancer associated fibroblasts (CAF) are present in DP, MS and IN LMEs each with distinct phenotypes. To functionally analyze the contribution of CAFs to PTCL maintenance, we established multiple patient-derived tumor xenografts (PDXs) that retain the same LME categories of primary tumors. We then tested lymphoma cell survival in co-cultures of CAFs and PTCL cells from matched (e.g., IN-CAF + IN-PTCL) and unmatched PDXs (e.g., IN-CAF + DP-PTCL). Matched co-cultures sustained lymphoma cell proliferation to a higher extent than unmatched, independently of LME subtype. This suggests that CAF are distinctly reprogrammed within each LME. We hypothesize that pro-tumoral CAF phenotypes require adaptive gene expression in coordination with lymphoma cells' phenotypes. RNA-Seq of matched IN-CAF co-cultured with IN-PTCL revealed activation of pathways like “Transcription”, “DNA replication” and “Translation” including the upregulation of CDK7. Although CDK7 plays a minor role in cell cycle progression, its phosphorylation of the RNA polymerase II as part of the TFIIH complex is critical in activating and coordinating “cell state”-defining transcription. To assess CDK7's role in establishing lymphoma-supportive CAF states, IN-PTCL PDX mice were treated with the covalent selective CDK7 inhibitor YKL-5-124 for 12 h followed by transcriptomic and proteomic profiling of IN-PTCL and IN-CAFs. In IN-CAFs, CDK7 inhibition altered gene and protein expression of several collagens and cytokines, including COL1A1, COL1A2, MMP8, and CXCL12. Ligand-receptor analysis identified CXCL12-CXCR4 as the key CDK7-dependent interaction between IN-CAFs and IN-PTCL cells. IN-PTCL cells showed activation of DNA damage repair pathways, including upregulation of ALYREF and EIF4E, likely as compensatory mechanisms for decreased CDK7-dependent transcription. Since these may represent acquired therapeutic vulnerabilities, we conducted viability screenings in co-cultures of IN-PTCL and IN-CAF isolated from YKL-5-124 (and vehicle) tretated PDX mice. Cells were subsequently exposed for 48 h, individually and in co-cultures, to a drug library containing 40 clinical-phase compounds. This approach led to the identification of 14 compounds with synergistic anti-lymphoma effects. Among these, ten drugs including the XPO1 inhibitor Selinexor, the HSP90 inhibitor PU-H71 and the translation inhibitor Omacetaxine exhibited enhanced anti-lymphoma activity specifically in the co-culture setting, whereas four compounds showed reduced efficacy. This was not seen in mismatched co-cultures and indicates a potential protective role of CAFs and, importantly, that this mechanism is CDK7 dependent. To address the limitations of PDX models established in immunodeficient mice with limited ability to capture the immune-mediated effects of drug treatments, we developed an IN-PTCL syngeneic model by intrasplenic implantation of the murine T-cell lymphoma cell line EL4. Treatment with YKL-5-124 significantly reduced the tumor burden compared to vehicles. Cytokine profiling (immunoblotting array) and ELISA of culture medium of YKL-5-124 treated EL4-CAFs showed reduced levels of pro-inflammatory cytokines such as CXCL12, IL6, CCL2, ICAM-1, and CXCL1 indicating that CDK7 is necessary in establishing pro-lymphomagenic IN-CAF phenotypes. In conclusion, CDK7 activity is required to sustain specific pro-tumoral crosstalk interactions of matched CAFs and PTCL cells, which can be exploited therapeutically.