JUN mediates glucocorticoid resistance by stabilizing HIF1a in T cell acute lymphoblastic leukemia.

Dexamethasone (Dex) plays a critical role in T-ALL treatment, but the mechanisms of Dex resistance are poorly understood. Here, we demonstrated that the expression of JUN was regulated in Dex-resistant T-ALL cell lines and patient samples. JUN knockdown increased the sensitivity to Dex. Moreover, the survival data showed that high expression of JUN related to poor prognosis of T-ALL patients. Then, we generated dexamethasone-resistant clones and conducted RNA-seq and ATAC-seq. We demonstrated that the upregulation of JUN was most significant and regulated by JNK pathway in Dex-resistant cells. High-throughput screening showed that HIF1α inhibitors synergized with Dex could enhance Dex resistance cells death in vitro and in vivo. Additionally, JUN combined and stabilized HIF1α in Dex resistance cells. These results reveal a new mechanism of Dex resistance in T-ALL and provide experimental evidence for the potential therapeutic benefit of targeting the JNK-JUN-HIF1α axis for T-ALL treatment.

ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

Cancer-cell-derived fumarate suppresses the anti-tumor capacity of CD8+ T cells in the tumor microenvironment.

Metabolic alterations in the microenvironment significantly modulate tumor immunosensitivity, but the underlying mechanisms remain obscure. Here, we report that tumors depleted of fumarate hydratase (FH) exhibit inhibition of functional CD8+ T cell activation, expansion, and efficacy, with enhanced malignant proliferative capacity. Mechanistically, FH depletion in tumor cells accumulates fumarate in the tumor interstitial fluid, and increased fumarate can directly succinate ZAP70 at C96 and C102 and abrogate its activity in infiltrating CD8+ T cells, resulting in suppressed CD8+ T cell activation and anti-tumor immune responses in vitro and in vivo. Additionally, fumarate depletion by increasing FH expression strongly enhances the anti-tumor efficacy of anti-CD19 CAR T cells. Thus, these findings demonstrate a role for fumarate in controlling TCR signaling and suggest that fumarate accumulation in the tumor microenvironment (TME) is a metabolic barrier to CD8+ T cell anti-tumor function. And potentially, fumarate depletion could be an important strategy for tumor immunotherapy.

The third-generation anti-CD30 CAR T-cells specifically homing to the tumor and mediating powerful antitumor activity.

CAR T-cell therapy is well tolerated and effective in patients with Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). However, even second- generation anti-CD30 CAR T-cells with CD28 (28z) costimulatory domains failed to achieve the desired rate of complete responses. In the present study, we developed second-generation (CD28z) and third-generation (CD28BBz) CAR T-cells targeting CD30 and investigated their efficacy in vitro and in vivo. Both of CD28z and CD28BBz anti-CD30 CAR T cells were similar regarding amplification, T cell subsets distribution, T cell activity, effector/memory and exhaustion. However, we found that the 28BBz anti-CD30 CAR T-cells persist long-term, specifically homing to the tumor and mediating powerful antitumor activity in tumor xenograft models. Subsequently, we also demonstrated that the third generation anti-CD30 CAR T-cells have miner side effects or potential risks of tumorigenesis. Thus, anti-CD30 CAR T-cells represent a safe and effective treatment for Hodgkin lymphoma.

Establishment of a New Cryopreservation Solution for Chimeric Antigen Receptor T Cells.

Preservation and transportation are essential for the clinical application of chimeric antigen receptor T (CAR-T) cells. This study aimed to optimize a cryopreservation solution for CAR-T cells and evaluate the antitumor efficiency of CAR-T cells using this optimized solution in vitro and in vivo. First, the stability of the cryopreservation solution for CAR-T infusion was detected by the L27 (37) orthogonal experiment. Subsequently, osmolality and pH were analyzed for the preservation reagent. Additionally, apoptosis and CAR expression of CAR-T cells were measured by flow cytometry, and the cytotoxicity was determined by calcein-AM staining. The results showed that cryopreservation solutions used in this study demonstrated high chemical stability, which induced only 2% CAR-T cells apoptosis in optimal solutions, which were slightly lower than other commercial solutions. Moreover, the CAR expression was not significantly affected by preservation with these solutions. There were no significant differences in the cytotoxicity between fresh and thawed CAR-T cells cryopreserved in the cryopreservation solutions in vivo and in vitro. This study developed a new cryopreservation solution for CAR-T cells, and it was safe and also had negligible effects on the CAR-T cells antitumor activity.

CAR T cells targeting CD99 as an approach to eradicate T-cell acute lymphoblastic leukemia without normal blood cells toxicity.

CAR T cell therapy has shown dramatic clinical success in relapsed or refractory B-ALL and other hematological malignancies. However, the loss of specific antigens, cell fratricide, T cell aplasia, and normal T cell separation are challenges in treating T cell leukemia/lymphoma with CAR T therapy. CD99 is a promising antigen to target T-ALL and AML as it is strongly expressed on the majority of T-ALL and AML. Here, we isolated a low-affinity CD99 (12E7) antibody, which specifically recognizes leukemia cells over normal blood cells. Moreover, T cells transduced with an anti-CD99-specific CAR that contained the 12E7 scFv expanded with minor fratricide and without normal blood cells toxicity. We observed that our anti-CD99 CAR T cells showed robust cytotoxicity specifically against CD99+ T-ALL cell lines and primary tumor cells in vitro and significantly prolonged cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) models survival in vivo. Together, our results demonstrate that anti-CD99 CAR T cells could specifically recognize and efficiently eliminate CD99+ leukemia cells.

Killing effect of NK92 cells modified with CD33-CAR on CD33+ acute myeloid leukemia cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To construct CD33-CAR modified NK92 cells based on CD33-scFv sequence, and to explore its killing effect on CD33+ AML (acute myeloid leukemia) cells. Methods: DNA fragment encoding CD33-CAR was synthesized by gene synthesis and molecular cloning technology and then cloned into lentiviral vector. Lentivirus were packaged and used to transfect NK92 cells. The transfection efficiency was detected by flow cytometry, and puromycin was used to screen NK92 cells stably expressing CD33-CAR (CD33-CAR-NK92). Killing effect of CD33-CAR-NK92 cells on AML cells in vitro was examined with calcein-AM release assays. IFN-γ secretions of NK92 cells and CD33-CAR-NK92 cells were measured by ELISA. Results: The pCDH-CD33-CAR lentiviral vector was successfully constructed. After lentiviral transfection, about 18.7% of NK92 cells express CD33-CAR (referred as CD33-CARNK92 cells). The percentage of CD33-CAR+ NK92 cells was about 86.3% after puromycin selection. In contrast to unmodified NK92 cells, significantly higher cytotoxic effect against CD33+ MOLM-13 cells was found in CD33-CAR-NK92 cells (P<0.01); however, there was no significant difference in cytotoxicity against CD33- JURKAT cells between NK92 cells and CD33-CAR-NK92 cells (P> 0.05). After co-culture at an effect-target ratio of 2∶1 for 6 hours, the level of IFN-γ secreted by the CD33-CAR modified NK92 cells was significantly higher than that of the unmodified ([190.97±11.52] vs [88.41±2.75]pg/ml, P<0.01). Conclusion: The CD33-CARNK92 cells could specifically recognize CD33 antigen and kill CD33+ AML cells in comparison with the unmodified NK92 cells, which provides experimental basis for clinical transformation of CD33-CAR-NK92 cells in treatingAML.