Natarajan, Amar

Introduction Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of clinically aggressive entities. ~30% of cases cannot be further classified and are designated as PTCL - not otherwise specified (NOS). We delineated two novel molecular subgroups among PTCL-NOS with distinct clinical and biological features: PTCL-GATA3 which is characterized by high expression of GATA3, a master regulator of T-helper-2-cell (TH2) differentiation, and its target genes (i.e., CCR4, IL4, IL13) and PTCL-TBX21 which is characterized by high expression of TBX21, a master regulator of TH1 differentiation, and its target genes (i.e., CXCR3, IFNG), with the GATA3 subtype showing significantly worse clinical outcomes (overall survival). Nonsense mutations in CCR4 are found predominately in PTCL-GATA3, often within the cytoplasmic domain and gain-of-function in nature. Methods Mutation and copy number data were generated from whole exome sequencing (TFH phenotype excluded following LLMPP pathology review; n = 153). Single nucleotide variants were determined using Mutect2 and Varsvan2 using in-house filtering. Copy number (CN) profiles were generated using CNVkit. Four CCR4 cytoplasmic domain mutants and controls (cytoplasmic domain knockout [CDKO], wild-type CCR4 overexpression (CCR4-WT), and empty vector [EV]) were transduced into healthy-donor CD4+ T cells (n = 2). Polarization studies were generated using TH1- or TH2-associated cytokine exposure. Surface plasmon resonance was used to determine the binding affinity of CCR4 proteins to CCL17 and CCL22. In silico docking studies were done through Schrödinger's BioLuminate. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was performed through co-incubation of NK92 (CD16 and CD56) and CCR4 edited primary CD4+ T-cells (stained with calcein AM) for 2 hours at a 5:1 NK:T cell ratio by 7-AAD flow cytometry. Results PTCL-GATA3 had a higher frequency of CN aberrations (e.g. TP53, PTEN CN loss) and a more aberrant genome (p < 0.001) than PTCL-TBX21, including mutations with genes associated with DNA repair and damage (TP53, ATM) and T-cell differentiation (CCR4). CCR4 mutations were virtually mutually exclusive with TP53 mutations, and these cases showed higher CCR4 mRNA and protein expression than CCR4-WT cases (p < 0.05). Primary CD4+ T-cells with ectopic expression of the CCR4 variants, especially CCR4-Q330X, did not require CD3/CD28 for proliferation, bypassing CD3/CD28 ligation for T-cell activation. In addition, proliferation rates were similar between TH2-conditional media and normal media, but proliferation and TBX21 expression in TH1-conditional media were inhibited, implying a novel role of CCR4 in T-cell receptor (TCR) activation and TH-differentiation. CCR4 variant proteins showed higher binding affinity for their natural ligands, CCL22 and CCL17, with mutants exhibiting tighter binding to both ligands (1.5- to 3-fold decrease in KD) by surface plasmon resonance and in silico modeling studies. The in silico models demonstrated mutant CCR4 bound mogamulizumab tighter than CCR4-WT, consistent with a higher ADCC-induced apoptosis rate, highest with CCR4-Q330X mutation. CCR4-Y331X exhibited a worse docking score compared to CCR4-WT and a lower ADCC-induced apoptosis rate. Conclusions Our findings reveal distinct genetic characterizations including aberrant TP53 and PI3K signaling and CCR4 mutations in PTCL-GATA3. These CCR4 mutations are shown to have a key role in PTCL-GATA3 pathogenesis by dysregulating TH-differentiation and aberrantly activating the TCR. CCR4 mutation promotes the TH2 phenotype by blocking TH1 differentiation. CCR4 mutant proteins demonstrated an increased binding affinity towards its known ligands, CCL17 and CCL22, and mogamulizumab, consistent with previous studies exhibiting mogamulizumab efficacy against CCR4 mutations. However, the most frequent CCR4 mutant, Y331X, showed worse binding compared to other CCR4 mutants or CCR4-WT, a mutation that showed Mogamulizumab-resistance in a prior CTCL study as well as in an in vitro ADCC assay. The results emphasize aberrant pathways implicated by genomic and clinical evaluation of two novel PTCL subtypes, as well as support the novel role of mutant CCR4 in enhancing T-cell lymphomagenesis and T-helper differentiation, highlighting multiple therapeutic targets.

Abstract Antibody-drug conjugates (ADCs) are increasingly being used in clinic, and many novel ADCs are being tested in clinical trials. Most ADCs act by delivering cytotoxic payloads into target cells. The activity of most ADCs is predicated upon internalization to release the cytotoxic payloads intracellularly to act on the malignant cells. The degree of ADC internalization is incomplete or absent for many cell surface targets, limiting the activity of ADCs. PROTACs are heterobifunctional molecules that result in the polyubiquitylation of their targets by inducing proximity with E3 ligases. The proteasome then recognizes the poly-ubiquitin chains and degrades these targets. We hypothesized that PROTACs that act on the same targets as ADCs could improve the internalization and efficacy of these ADCs. Using multiple cell lines derived from breast, lung or pancreatic cancers that express the oncogenic cell surface proteins EGFR (ERBB1), HER2 (ERBB2) or MET (HGF), we measured antibody internalization with live cell imaging. Antibodies were labeled with a pH sensitive reagent that fluoresces red (640 nm) upon exposure to acidic environments. Fluorescence was measured over time to create an area under the curve which was compared between the cell lines that were treated with and without PROTACs. Antibody internalization was observed in all cell lines that expressed the targets recognized by the antibodies. The addition of PROTACs targeting the same oncogenic cell surface proteins as the antibodies significantly increased antibody internalization beyond that observed without PROTACs. Internalization of antibodies was confirmed by measuring intracellular immunoglobulins by Western blot. Antibody internalization with PROTACs was dose-dependent, increased over time and was blocked by either the clatharin inhibitor Dyngo4a or the proteasome inhibitor MG132. In the case of HER2 for which there are FDA-approved anti-HER2 ADCs, the cytotoxicity of trastuzumab emtansine (TDM1) significantly increased with the addition of a HER2-targeting PROTAC across a range of doses. PROTACs that target the same oncogenic cell surface proteins as antibodies improve antibody internalization and ADC cytotoxicity, in a process dependent upon clatharin and the proteasome. This novel application of PROTACs may impact the use of ADCs, and provides a rationale to combine these agents in clinical trials. Citation Format: Ezequiel J. Tolosa, Lin Yang, Jennifer Ayers-Ringler, Jayapal Mallareddy, Janet Schaefer-Klein, Farhad Kosari, Amar Natarajan, Aaron S. Mansfield. Proteolysis targeting chimera (PROTAC)-driven internalization of antibodies targeting oncogenic cell surface receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4518.