TP63 Fusions Drive Enhancer Rewiring, Lymphomagenesis, and Dependence on EZH2

Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas, which is highly unique among chromosomal translocations. Specifically, TP63 rearrangements were found in 5-10% of ALK-negative anaplastic large cell lymphoma (ALK- ALCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and primary cutaneous ALCL, 7% of mycosis fungoides with large cell transformation (MF-LCT), and 1% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. This contrasts with rearrangements of DUSP22, which are also observed in ALK- ALCLs and confer a good prognosis. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients.

Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 rearrangements suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell development and lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice, which results in expression beginning during early lymphoid development. Transgenic mice had both cytokine and transcriptional evidence of immune activation as well as T cell repertoire narrowing, consistent with clonal selection. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma that mimicked ALCL, PTCL-NOS and MYC-expressing DLBCL. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR screening, transcriptomic, epigenomic, and proteomic analyses. We also performed a domain-focused CRISPR-Cas9 knockout scan using 452 sgRNAs tiling the entire coding regions of TBL1XR1 and TP63 to clarify essential oncogenic domains within TBL1XR1-TP63. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions (TBL1XR1, FOXK2, etc.) interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 fusions (which recapitulates the deltaNp63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of EZH2, which encodes the histone H3K27 methylase. Knockdown of the TP63 fusion in TCL cell lines led to complete loss of H3K27me3. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 inhibitor tazemetostat and the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, was highly effective in three different in vivo models of TP63-rearranged TCL. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2.

Wu:Daiichi Sankyo: Research Funding. Liu:Astra Zeneca: Current Employment. Jain:Abcuro, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Crispr Therapeutics: Membership on an entity's Board of Directors or advisory committees; Acrotech LLC: Research Funding; Myeloid Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Mersana Therapeutics: Research Funding; SecuraBio: Membership on an entity's Board of Directors or advisory committees; SIRPant Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jones:Astra Zeneca: Current Employment. Stevenson:Novartis: Current Employment. Louissaint:Lymphoma Research Foundation: Research Funding. Meyerson:Janssen: Research Funding; Labcorp: Other: other support; Ono: Research Funding; Isabel: Consultancy; Interline: Consultancy; Bayer: Consultancy, Research Funding. Adelman:Syros Pharmaceuticals: Consultancy; CAMP4 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Brown:Novartis: Consultancy, Research Funding; Kronos Bio: Membership on an entity's Board of Directors or advisory committees; H3 Biomedicine: Membership on an entity's Board of Directors or advisory committees; GV20 Oncotherapy: Membership on an entity's Board of Directors or advisory committees. Weinstock:Bantam: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Secura: Research Funding; Abcuro: Research Funding; Travera: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Merck: Current Employment; Ajax: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Research Funding.