Targeting cytohesin-1 suppresses acute myeloid leukemia progression and overcomes resistance to ABT-199

Abstract

Adhesion molecules play essential roles in the homeostatic regulation and malignant transformation of hematopoietic cells. The dysregulated expression of adhesion molecules in leukemic cells accelerates disease progression and the development of drug resistance. Thus, targeting adhesion molecules represents an attractive anti-leukemic therapeutic strategy. In this study, we investigated the prognostic role and functional significance of cytohesin-1 (CYTH1) in acute myeloid leukemia (AML). Analysis of AML patient data from the GEPIA and BloodSpot databases revealed that CYTH1 was significantly overexpressed in AML and independently correlated with prognosis. Functional assays using AML cell lines and an AML xenograft mouse model confirmed that CYTH1 depletion significantly inhibited the adhesion, migration, homing, and engraftment of leukemic cells, delaying disease progression and prolonging animal survival. The CYTH1 inhibitor SecinH3 exerted in vitro and in vivo anti-leukemic effects by disrupting leukemic adhesion and survival programs. In line with the CYTH1 knockdown results, targeting CYTH1 by SecinH3 suppressed integrin-associated adhesion signaling by reducing ITGB2 expression. SecinH3 treatment efficiently induced the apoptosis and inhibited the growth of a panel of AML cell lines (MOLM-13, MV4-11 and THP-1) with mixed-lineage leukemia gene rearrangement, partly by reducing the expression of the anti-apoptotic protein MCL1. Moreover, we showed that SecinH3 synergized with the BCL2-selective inhibitor ABT-199 (venetoclax) to inhibit the proliferation and promote the apoptosis of ABT-199-resistant leukemic cells. Taken together, our results not only shed light on the role of CYTH1 in cell-adhesion-mediated leukemogenesis but also propose a novel combination treatment strategy for AML.

Keywords: ABT-199; CYTH1; MCL1; SecinH3; acute myeloid leukemia; cell adhesion.

Fig. 1. Aberrant expression of CYTH1 in AML is associated with poor prognosis.

a CYTH1 mRNA expression was analyzed in primary AML cells (n = 173) and normal bone marrow (NBM) cells (n = 70) from the GEPIA database. *P < 0.05. b Analysis of CYTH1 mRNA expression in primary AML samples (n = 179) from TCGA database, categorized into FAB subgroups. c Analysis of CYTH1 mRNA expression in AML samples with various genetic abnormalities and normal hematopoietic progenitors from the BloodSpot database. d Immunoblots of CYTH1 in NBM cells, primary patient AML cells, and AML cell lines, as indicated. HSP90 served as a loading control. e Kaplan–Meier survival curves of AML patients from TCGA database with high CYTH1 mRNA expression (n = 90) and low CYTH1 mRNA expression (n = 89). f Univariate and multivariate Cox proportional hazard analyses revealed that CYTH1 expression was associated with OS in TCGA AML cohort. The multivariate model was additionally adjusted for TP53, DMNT3A, RUNX1, and FLT3 mutations. CI confidence interval, WBC white blood cell count.

Fig. 2. CYTH1 is required for AML adhesion and migration in vitro.

a U937 and MV4-11 cells were transduced with lentiviruses expressing shRNA targeting control (shCtrl) or CYTH1 (sh1 and sh2). CYTH1 knockdown efficiency was analyzed by qRT-PCR (upper) and immunoblotting (bottom). b–c U937 and MV4-11 cells were transduced with CYTH1 shRNAs from a GFP-expressing vector and used in the adhesion assay. Flow cytometry analysis was used to determine the normalized absolute numbers of adherent GFP⁺ leukemic cells (b). Representative images of the adhesion of GFP⁺ leukemic cells (with or without CYTH1 knockdown) to mesenchymal stem cells (MSCs) (left) and quantification of adhered cells (right) (c). Scale bar, 100 μm. d Results from the in vitro migration assay using CYTH1-depleted or -undepleted AML cells. Representative images (left) and quantification (right) of cell migration in the transwell culture system. Scale bar, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. CYTH1 is required for AML homing and engraftment in vivo.

a Graphic illustration of in vivo leukemia homing and progression analysis in an AML xenograft mouse model. Human AML U937 cells expressing the GFP marker were treated with shRNA targeting CYTH1 mRNA (shCYTH1 [sh1]) or the control shRNA (shCtrl). GFP⁺ cells were injected into irradiated NPG mice to assess AML homing to bone marrow (BM) 24 h post-injection and monitor AML progression every 7 days. b Flow cytometry analysis of human CD45 (hCD45) expression in AML cells (with or without CYTH1 knockdown) in the BM of recipient mice 24 h post-injection (n = 5 per group). c Percentages of GFP⁺ cells in the peripheral blood (PB) of mice at the indicated time points post-injection (n = 4 per group). d, e Percentages of GFP⁺ cells in the BM and spleens of mice 14 days post-injection (n = 4 per group). f Kaplan–Meier survival curves of U937 xenografts (n = 4 per group). **P < 0.01; ***P < 0.001.

Fig. 4. SencinH3 attenuates AML cell growth by inhibiting adhesion and survival programs.

a, b U937 cells were treated with SecinH3 at the indicated concentrations for 2 h, prior to cell adhesion (a) and migration (b) analysis. c Immunoblots showing the expression of ITGB1, ITGB2, MMP2, and MMP9 in U937 cells treated with SecinH3 (left) and transduced with CYTH1 shRNAs (sh1 and sh2) (right). Actin served as a loading control. d, e Flow cytometry analysis of active ITGB2 (d) and CXCR4 (e) expression on the surface of U937 and MV4-11 cells treated with SecinH3 and transduced with CYTH1 shRNAs (sh1). MFI mean fluorescence intensity. f Flow cytometry analysis of apoptosis in MLL-rearranged (MV4-11, MOLM-13, and THP-1) and non-MLL-rearranged (U937, KG1, and HEL) AML cell lines treated with SecinH3 at the indicated concentrations for 48 h. g MV4-11, MOLM-13, and THP-1 cells were treated with SecinH3 at various concentrations for the indicated times before their relative cell viability was assessed using the CCK-8 assay. h Flow cytometry analysis of control and CYTH1-overexpressing (CYTH1-OE) MV4-11 cell apoptosis after treatment with SecinH3 (50 μM) for 48 h. i Immunoblots of cleaved Caspase-3, MCL1, and BCL-2 expression in MV4-11 (left) and MOLM13 cells (right) treated with SecinH3. HSP90 served as a loading control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5. SecinH3 inhibits the growth of AML xenograft tumors.

a Tumor volume in each group was assessed by calipers and calculated at the indicated time points post-treatment. b Representative images of the tumor burden of the Vehicle or SecinH3 groups after 12 days of treatment. c Images of tumors (left) and tumor weights (right) in each group after 12 days of treatment. d, e Immunohistochemistry analysis of Ki-67, cleaved Caspase-3 (C-Casp3), MMP2, and MMP9 expression in each treatment group. Representative images (d) and quantification of immunostained areas (e) are shown. Scale bar, 20 μm. *P < 0.05; **P < 0.01.

Fig. 6. SecinH3 sensitizes AML cells to ABT-199.

a Cell viability of MV4-11 and MOLM-13 cells was measured using the CCK-8 assay at 48 h after treatment with SecinH3, ABT-199, or a combination of both. Contour plots of synergy scores generated from the cell viability dose matrix of SecinH3 and ABT-199 using the zero interaction potency (ZIP) model. The optimal concentration range for drug synergy is shown in the white dashed box. ZIP scores were analyzed using SynergyFinder (https://synergyfinder.fimm.fi/synergy/synfin_docs/). b Flow cytometry analysis of apoptosis in MV4-11 (upper) and MOLM-13 (bottom) cells treated with SecinH3 (12.5 μM), ABT-199 (25 nM), or a combination of both (Combo) for 48 h. c Graphical illustration of MOLM-13 luciferase AML xenograft generation and treatment strategy. Treatment started on day 7 post-engraftment and was administered every other day as illustrated. d Representative images (left) and quantification (right) of leukemic burden, as assessed by luciferase luminescence signals in the xenografts (n = 3 per group). e Representative images of mouse spleens are shown above, with their weights plotted below (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. SecinH3 overcomes resistance to ABT-199 by modulating MCL1 expression.

a Drug matrix of SecinH3 and ABT-199 for ABT-199-resistant MV4-11 (MV4-11-R) cells treated for 48 h. Synergy was assessed using ZIP scores. b Flow cytometry analysis of MV4-11-R apoptosis after treatment with SecinH3 (12.5 μM), ABT-199 (200 nM or 400 nM), or a combination of both (Combo) for 48 h. ***P < 0.001. c Immunoblots of MCL1 and BCL2 expression in MV4-11-R (left) and control MV4-11 (right) cells treated with SecinH3 (12.5 μM), ABT-199 (200 nM for MV4-11-R and 25 nM for MV4-11) or a combination of both (Combo) for 48 h. Actin served as a loading control.