North American Adult T-Cell Leukemia Lymphoma Has Frequent Mutations in CCR4 and Responds in Vitro to a Small Molecule CCR4 Antagonist

Background:

Adult T-cell leukemia/lymphoma (ATLL) is a fatal cancer caused by the human T-lymphotropic virus-1 (HTLV-1) and somatic mutations in virus-infected CD4 T cells. With limited treatment options, patients with ATLL urgently need new and improved treatment strategies. CCR4 encodes a chemokine receptor that is mutated in a third of ATLL patients diagnosed in Japan (J-ATLL), but its mutation status and expression pattern have not been reported for ATLL cases in North America (NA-ATLL). Furthermore, a CCR4-targeting therapy, Mogamulizumab, was reported to have clinical efficacy in J-ATLL but not NA-ATLL patients although the underlying mechanism is unknown. This study aims to address these issues and to evaluate the preclinical activity of an oral small molecule CCR4 antagonist that effectively blocks the binding of the two CCR4 ligands, CCL17 and CCL22.

Tivumecirnon (formerly known as FLX475) is a small molecule CCR4 antagonist currently in a Phase 1/2 study (NCT03674567) in several types of advanced cancer including EBV+ NK/T cell lymphoma. Favorable clinical antitumor and biomarker activity has been reported thus far.

In this study, we used CCR4-351, a CCR4 antagonist that is a close structural analog of tivumecirnon.

Methods:

CCR4 mutation status was determined based on bulk RNA-seq (n=10) and whole genome sequencing analysis (n = 10). CCR4 ligands concentration in patient plasma and culture supernatants were determined by ELISA. Flow cytometry was used to compare cell surface CCR4 expression on normal (n=4) and ATLL (n=7) CD4 T cells. Flow cytometry is also used to evaluate the expression pattern of the two CCR4 conformers, R1 (responding to both CCL17 and CCL22) and R2 (responding to CCL22 only), as well as ligand-induced CCR4 endocytosis. The ability of CCR4-351 to block chemotaxis of ATLL cells was studied using trans-well assays.

Results:

Eight out of 17 NA-ATLL cases (47.1%) carried CCR4 gene alterations including in-frame and frameshift indels, as well as nonsense mutations targeted to the cytoplasmic tail and the trans-membrane domain. Thus, CCR4 is possibly mutated at an even higher rate in NA-ATLL compared to J-ATLL. Circulating ATLL cells (CD4⁺CD7^-CD8^-) express markedly increased levels of CCR4 relative to healthy control CD4 T cells. A minority of NA-ATLL plasma samples contain abnormally elevated CCL17 (2/17) and CCL22 (4/17) levels. When cell culture supernatants were examined, we found that NA-ATLL but not J-ATLL cell lines can produce these two chemokines. Specifically, NA-ATLL cell lines produced CCL17 and CCL22 at 0.06-2.5 ng/mL/e6 cells and 0.35–3.9 ng/mL/e6 cells, respectively, compared to 0.014-0.033 ng/mL/e6 cells and 0.019-0.127 ng/mL/e6 cells for J-ATLL cell lines. In transwell assays, both CCL17 and CCL22 elicited strong chemotaxis activity in the two J-ATLL cell lines tested. Unexpectedly, 3 out of 4 NA-ATLL cell lines responded only to CCL22 but not CCL17 despite very high levels of cell surface CCR4 measured by either mean fluorescence intensity of R1 or R1/R2 ratio. It has been established in the literature that CCR4 signaling is biased towards CCL22 in that CCL22 acts as a full agonist while CCL17 is a partial agonist. We indeed observed this bias in ligand-mediated endocytosis assays with CCR4 WT cell lines but this bias was lost in CCR4 mutated cell lines due to reduced endocytosis triggered by CCL22. Lastly, the small molecule CCR4 antagonist, CCR4-351 exhibited potent chemotaxis inhibitory activity with IC₅₀ values ranging from 7–60 nM. CCR4-351 treatment also caused a mild reduction in cell proliferation, suggesting additional anti-ATLL mechanism of action.

Conclusion:

In summary, in this first study of the CCR4 pathway status in NA-ATLL, we found that nearly half of the cases in our ATLL cohort carried a mutated CCR4 gene. Contrary to primary ATLL cells, ATLL cell lines exhibit a wide variation in CCR4 expression. Gain-of-function CCR4 mutations correlated with reduced receptor endocytosis triggered by exogenous CCL22. CCL22 but not CCL17 induced strong chemotaxis in NA-ATLL cell lines, which can be potently inhibited by a small molecule CCR4 antagonist, CCR4-351. Since extramedullary presentation is frequently seen in NA-ATLL and central nervous system involvement is an adverse prognostic feature, inhibiting chemotaxis with a CCR4 antagonist such as tivumecirnon may be an effective therapeutic approach for this devastating disease.