Repressing HIF-1α-induced HDAC9 contributes to the synergistic effect of venetoclax and MENIN inhibitor in KMT2Ar AML

Abstract

KMT2A-rearranged acute myeloid leukemia (KMT2Ar-AML) is an aggressive subtype of AML with poor response and prognosis. KMT2Ar-AML has been demonstrated to be sensitive to BCL2 inhibitor venetoclax (VEN), but these patients are unable to benefit from current VEN-based regimen (VEN plus azacitidine or low dose-cytarabine), so a novel and KMT2A rearrangement-specific targeting partner is required, and MENIN inhibitor (MEN1i) is a promising one. Herein, we investigated the effect and mechanism of VEN plus MEN1i in KMT2Ar-AML. Our results showed that VEN and MEN1i exhibited a striking synergistic effect in KMT2Ar-AML cell lines (in vitro), primary KMT2Ar-AML cells (ex vivo), and MOLM13 xenotransplantation model (in vivo). Furthermore, we found that VEN plus MEN1i significantly enhanced apoptotic induction in KMT2Ar-AML cell lines. VEN or MEN1i monotherapy disrupted balance of BCL-2/BCL-XL or down-regulated HOXA9/MEIS1, respectively, but these mechanisms were not further strengthened by their combination. RNA-Sequencing identified that HDAC9 was specifically repressed by VEN plus MEN1i rather than monotherapy. We demonstrated that HDAC9 was indispensable for KMT2Ar-AML proliferation and its repression contributed to proliferation inhibition of VEN plus MEN1i. Moreover, we found that hypoxia induced HDAC9 expression in KMT2Ar-AML, and VEN plus MEN1i inhibited hypoxia pathway, especially HIF-1A, and its target HDAC9. As our results indicated, VEN plus MEN1i-mediated HDAC9 down-regulation was partially dependent on HIF-1A repression in KMT2Ar-AML. Hypoxia induction sensitized KMT2Ar-AML to VEN plus MI-503-mediated proliferation inhibition and apoptosis induction. Therefore, repressing HIF-1A-induced HDAC9 contributed to the synergistic effect of VEN and MEN1i in KMT2Ar-AML.

Keywords: Acute myeloid leukemia; HIF-1A-induced HDAC9; KMT2A rearrangement; MENIN inhibitor; Venetoclax.

Fig. 1

VEN and MEN1i synergistically inhibited KMT2Ar-AML proliferation. A IC50 of VEN in different AML cell lines (72 h); B IC50 of MI-503 in different AML cell lines (7 days); C Growth inhibition and synergistic index (combination index [CI] was calculated via CalcuSyn software) in THP-1 (expressing KMT2A-MLLT3), MV4-11 (expressing KMT2A-AFF1), and MOLM13 (expressing KMT2A-MLLT3) cells after treatments with different concentrations of VEN, MI-503, and their combination (72 h); D Apoptotic induction (Annexin V-positive cells) was determined after treatments with VEN, MI-503, and their combination (72 h) [two-tailed Student's t-tests; **P < 0.01, ***P < 0.001]; E Growth inhibition and synergistic index of VEN plus MI-503 in primary bone marrow MNCs from KMT2Ar-AML patients (72 h); F Detecting the progression of tumor burden via fluorescence imaging after treating the CDX mouse model constructed by MOLM13-Luciferase with DMSO, VEN, MI-503 and their combination. G The relative luminescence unit (RLU) value was calculated for indicating leukemic burdens of treated mice (28 days) [two-tailed Student's t-tests; *P < 0.05, **P < 0.01, ***P < 0.001]. H Kaplan–Meier survival curves in the MOLM13 xenotransplantation model [two-tailed Student's t-tests; *P < 0.05, **P < 0.01, ***P < 0.001]

Fig. 2

VEN plus MEN1i impaired KMT2Ar-AML growth via repressing HIF-1A-induced HDAC9. A After treating KMT2Ar-AML cell lines THP-1, MV4-11, MOLM13 with DMSO, VEN, MI-503 and their combination for 72 h, RNA sequencing was performed and differentially expressed genes (DEGs) were analyzed; Overall strategy for screening key DEGs related to the synergistic mechanism of VEN plus MI-503 in KMT2Ar-AML was shown; Specific DEGs in VEN plus MI-503 group vs. DMSO group shared by three KMT2Ar-AML cell lines were listed; B The mRNA expression of HDAC9 in THP-1, MV4-11, and MOLM13 after treatments with DMSO, VEN, MI-503, and their combination [two-tailed Student's t-tests; **P < 0.01, ***P < 0.001 ****P < 0.0001]; C The protein expression of HDAC9 and HIF1A in THP-1, MV4-11, and MOLM13 after treatments with DMSO, VEN, MI-503 and their combination; D IC50 of TMP-269 in THP-1, MV4-11, MOLM-13, U937, HL-60, Kasumi-1, and OCI-AML3 cells (72 h); E Growth inhibition was mediated by HDAC9 knockdown in THP-1, MV4-11, and MOLM13 cells; F Different therapeutic effects of VEN plus MI-503 in normal control or HDAC9 knockdown THP-1, MV4-11, and MOLM13 cells were exhibited; Relative absorbance value (optical density [OD] ratio) to normal control for each group was presented and the difference value of OD ratio between two groups was marked as numbers between the histogram bars; G Hypoxia pathway gene set was enriched in the VEN plus MI-503 group compared to DMSO group, the horizontal axis represents the p-value, the vertical axis represents the standard enrichment fraction (NES), the size of the circle represents the -log (p adjusted) value, and different colors represent different gene sets; H The protein expression of HDAC9 and HIF1A in THP-1, MV4-11, and MOLM13 treated with DMOG. I Under HIF-1A inhibitor BAY 87-2243 treatment, HIF-1A mRNA (left) and protein (middle) levels were evaluated. Furthermore, HDAC9 mRNA was detected in DMSO or VEN (400 nM) plus MI-503 (700 nM)-treated THP1 cells with or without BAY 87-2243 3-day pre-treatment (72 h) [two-tailed Student's t-tests; *P < 0.05, **P < 0.01, ***P < 0.001]. J Under normoxia and hypoxia, THP-1 cells were treated by DMSO or VEN plus MI-503, and cell viability and cell apoptosis were evaluated [two-tailed Student's t-tests; **P < 0.01, ***P < 0.001 ****P < 0.0001]