Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models

Abstract

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.

Fig. 1

Genetic landscapes of TCL models identify targetable vulnerabilities. a Mutational characteristics of the most recurrently altered genes in patients, PDX models and cell lines of different subtypes of T- and NK-cell lymphomas. Highlighted in red is the frequency of recurrent mutations in different subtypes of T- and NK-cell lymphomas, based on published sequencing studies (Supplementary Table 3) with n indicating the number of cases included. b GISTIC summary plots of the significant copy number gains (left panel, red) and losses (right panel, blue) in 20 T-cell lymphoma cell lines. Y-axis: chromosomal position, X-axis: false-discovery rate (FDR) q values. c A subset of selected fusions in PDX models and cell lines, identified by RNA-seq. d Immunoblotting in ALCL cell lines for the indicated targets. Wild-type JAK2 is approximately 130 kDa and PCM-JAK2 is approximately 260 kDa. e Copy number variants of JAK1 and JAK2 corresponding to d. f IC50 values for ruxolitinib corresponding to d. g Workflow of the ruxolitinib in vivo trial. h Spleens were harvested from mice treated with vehicle or ruxolitinib. Spleen weight, infiltration of the spleens by hCD45/hCD2⁺ cells and AnnexinV/7-AAD staining of vehicle vs ruxolitinib treated mice. Statistics: Unpaired two-sided t-test, * p < 0.05. i Immunoblotting of MAC2A cells treated in vitro with DMSO or 1 μM ruxolitinib for 24 h and DFTL-28776 treated in vivo with vehicle or ruxolitinib for 7 days. Bar graphs in f and i indicate mean values of at least two independent experiments with error bars indicating standard error of the mean

Fig. 2

Genome-scale vulnerability screens reveal unique and common targets. a T-cell lymphoma specific vulnerabilities, ranked by Z-score and recurrence. b Corresponding false-discovery rate (FDR) q-values. c Corresponding gene expression level; RPKM, reads per kilobase of transcript, per million mapped reads. d Combined gene expression level and FDR. e Dependency score of MDM2 in TP53 -wild-type versus TP53-mutated cell lines. Comparison is by two-sided t-test with Welch correction. f MDMX dependency score across 391 cancer cell lines with TCL lines highlighted in red. g Knockdown of MDM2 and MDMX in TP53-wild-type cell lines KI-JK and SUP-M2 and TP53-mutated cell line SMZ-1 using GFP-expressing, doxycycline inducible shRNA. Data points indicate mean values with error bars indicating standard error of the mean

Fig. 3

Targeting MDMX and MDM2 with ALRN-6924 a Copy number variants and RNA expression levels of MDM2, MDMX, and TP53 across 21 T- and NK-cell lymphoma cell lines. b Protein levels of MDM2, MDMX and p53 by immunoblotting, corresponding to a. c TP53 mutation status, corresponding to a. d IC50 values of ALRN-6924 and RG-7112, corresponding to a. Bar graphs indicate mean values of at least two independent experiments performed in quadruplicates with error bars indicating standard error of the mean. Statistics: Unpaired two-sided t-test, *p < 0.05, **p < 0.01. e Tumor burden at all involved sites for each mouse. Tumor involvement is represented as % tumor weight per body weight for subcutaneous (s.c.) tumors, % spleen per body weight and % infiltration of hCD45/hCD2⁺ cells for bone marrow (BM) and peripheral blood (PB). Liver involvement is presented in grams, assessed by liver weight x % infiltration of hCD45/hCD2⁺ cells. Comparisons are by two-sided t-test with Welch correction. Error bars indicate standard error of the mean. f PET-CT scan showing axillary lymph node involvement with AITL at two sites (circled) prior to treatment with ALRN-6924 and complete remission after cycle 6