Acute myeloid leukemia (AML) is a high-mortality malignancy with poor outcomes. Azacitidine induces cell death and demonstrates treatment effectiveness against AML. Selinexor (KPT-330) exhibited significant benefits in combination with typical induction treatment for AML patients. Here, we explore the antitumor effect of KPT-330 combined with AZA in AML through CCK-8, flow cytometry, RT-qPCR, western blot, and RNA-seq. Our results showed that KPT-330 combined with AZA synergistically reduced cell proliferation and induced apoptosis in AML primary cells and cell lines. Compared to the control, the KPT-330 plus AZA down-regulates the expression of XPO1, eIF4E, and c-MYC in AML. Moreover, the knockdown of c-MYC could sensitize the synergy of the combination on suppression of cell proliferation and promotion of apoptosis in AML. Moreover, the expression of XPO1 and eIF4E was elevated in AML patient cohorts, respectively. XPO1 and elF4E overexpression was associated with poor prognosis. In summary, KPT-330 with AZA exerted synergistic effects by suppressing XPO1/eIF4E/c-MYC signaling, which provided preclinical evidence for further clinical application of the novel combination in AML.

Background More effective targeted therapy and new combination regimens are needed for Acute myeloid leukemia (AML), owing to the unsatisfactory long-term prognosis of the disease. Here, we investigated the synergistic effect and the mechanism of a histone deacetylase inhibitor, Chidamide in combination with Cladribine, a purine nucleoside antimetabolite analog in the disease. Methods Cell counting kit-8 assays and Chou-Talalay’s combination index were used to examine the synergistic effect of Chidamide and Cladribine on AML cell lines (U937, THP-1, and MV4-11) and primary AML cells. PI and Annexin-V/PI assays were used to detect the cell cycle effect and apoptosis effect, respectively. Global transcriptome analysis, RT-qPCR, c-MYC Knockdown, western blotting, co-immunoprecipitation, and chromatin immunoprecipitation assays were employed to explore the molecule mechanisms. Results The combination of Chidamide with Cladribine showed a significant increase in cell proliferation arrest, the G0/G1 phase arrest, and apoptosis compared to the single drug control in AML cell lines along with upregulated p21Waf1/Cip1 expression and downregulated CDK2/Cyclin E2 complex, and elevated cleaved caspase-9, caspase-3, and PARP. The combination significantly suppresses the c-MYC expression in AML cells, and c-MYC knockdown significantly increased the sensitivity of U937 cells to the combination compared to single drug control. Moreover, we observed HDAC2 interacts with c-Myc in AML cells, and we further identified that c-Myc binds to the promoter region of RCC1 that also could be suppressed by the combination through c-Myc-dependent. Consistently, a positive correlation of RCC1 with c-MYC was observed in the AML patient cohort. Also, RCC1 and HDAC2 high expression are associated with poor survival in AML patients. Finally, we also observed the combination significantly suppresses cell growth and induces the apoptosis of primary cells in AML patients with AML1-ETO fusion, c-KIT mutation, MLL-AF6 fusion, FLT3-ITD mutation, and in a CMML-BP patient with complex karyotype. Conclusions Our results demonstrated the synergistic effect of Chidamide with Cladribine on cell growth arrest, cell cycle arrest, and apoptosis in AML and primary cells with genetic defects by targeting HDAC2/c-Myc/RCC1 signaling in AML. Our data provide experimental evidence for the undergoing clinical trial (Clinical Trial ID: NCT05330364) of Chidamide plus Cladribine as a new potential regimen in AML.

IntroductionThis study aims to evaluate the efficacy and safety of the novel combination of Aza and HIA as the frontline induction therapy in newly diagnosed AML patients eligible for intensive chemotherapy (IC) (registered on ClinicalTrials.gov, number NCT04248595).MethodsAza (75mg/m2/d on days1-5 subcutaneous) is administered in combination with HIA [HHT 2mg/m2/d on days 4-8 intravenous over 3 hours, idarubicin 6mg/m2/d on days 4-6 intravenous, and cytarabine 100mg/m2/d on days 4-10 intravenous]. The primary endpoint was complete remission (CR) or CR with incomplete blood count recovery (CRi). Secondary endpoints were overall survival (OS), relapse-free survival (RFS), and adverse events (AEs).ResultsA total of 20 AML patients (aged 18-70 years) were enrolled between Jan 2020 and Sep 2022. 95% (19/20) of patients achieved CR/CRi, and 89.5% (17/19) had undetectable MRD, in which 94.7% (18/19) reached CR/CRi, and 88.9% (16/18) obtained MRD negative after the 1st cycle of induction therapy. Median OS and RFS were both not reached during the follow-up. The estimated 2-year OS and RFS were 87.5% (95%CI, 58.6% to 96.7%) and 87.1% (95%CI, 57.3% to 96.6%), respectively. No patient discontinued the treatment for AEs.DiscussionThis study provides preliminary evidence for this novel combination therapy as the first-line induction therapy for young or older AML patients fit for IC.

Liquid biopsy has been experimented with to identify the mutation of lymphoma based on next‐generation sequencing (NGS). We applied NGS analysis to circulating tumor DNA (ctDNA) in 20 lymphoma patients. Then, we compared treatment outcomes, and clinical characteristics among these patients, then investigated mutational profiling. Two independent cohorts of 241 patients with mature B cell lymphoma in Mature B‐cell malignancies data set (MBN) data set and 50 diffuse large B‐cell lymphoma (DLBCL) patients in DLBCL data set, were used to examine the association between gene mutations and prognosis. We found ctDNA positive group had significantly more relapsed/PD (7/12, 58.3%) and less CR/PR patients (1/12, 8.3%) compared to negative group (0, 0%) (5/8, 62.5%) (p < 0.001). Somatic alterations were identified in 12 of 20 patients and the total 11 mutations were: Ataxia telangiectasia mutated (ATM), TP53, BCL2, BTG2, CD28, EP300, IDH2, IRF8, JAK3, NOTCH1, and NRAS. ATM (S2168L) was found in SLL and TLBL for the first time. BTG2 (c.292_293del), CD28 (P119T), IRF8 (E74D) and NOTCH1 (c.4348 G > A) were newly detected in DLBCL, angioimmunoblastic T‐cell lymphoma, primary central nervous system lymphoma, and BCL for the first time respectively. We also disclosed an unreported mutation EP300 (c.1058_1059insC) in DLBCL. Our cases implied ctDNA detection consistent with the FISH of tissue samples to some extent, speculating new molecular subtypes of DLBCL, finding some potential drug‐resistant mutations, and suggesting disease recurrence. Moreover, in MBN and DLBCL datasets, patients with TP53 mutation had a significantly shorter OS (all p < 0.05) in both circulating free DNA and tumor tissue. The mutations (no SNP) of NOTCH1 (all p < 0.05) significantly contributed to worse OS in the two cohorts.

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