Aneta Baran

Effects of velcrin treatment on SLFN12 protein levels, PDE3A/B binding, and induction of cell death. A, HEL cells were treated with vehicle control (DMSO) or with the indicated doses of BAY 2666605 for 4 or 8 hours. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. GAPDH is shown as a loading control. B,SLFN12 mRNA expression of HEL cells after BAY 2666605 treatment at the indicated concentrations for 4 hours (left) or 8 hours (right). SLFN12 expression was assessed by qRT-PCR normalized to GAPDH and DMSO was used as a vehicle control (indicated by the dotted line). Data represent means ± SEM of three independent experiments, each conducted in triplicate. At each concentration, statistical significance was determined using a one-sample Student t test compared with 100%; ns, not significant. C, mRNA expression of respective genes in HEL cells transfected with a non-targeting siRNA control (indicated by the dotted line) and siRNA targeting PDE3A (left) or PDE3B (right). Data are expressed as the percentage of respective mRNA expression over non-targeting siRNA control (%) and represent means ± SEM of three independent experiments, each conducted in triplicate. Statistical significance was determined using a one-sample Student t test compared with 100%; ns, not significant, **, P ≤ 0.01. D, Immunoblot analysis of HEL cells. HEL cells were transfected with respective siRNA for 24 hours (see panel C), followed by 8 hours of low (1 μmol/L) or high (10 μmol/L) doses of BAY 2666605, using DMSO as a vehicle control. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies (E). HEL (left) or U937 (right) cells were treated with vehicle control (DMSO) or with low (1 μmol/L) or high (10 μmol/L) concentrations of BAY 2666605 for 24 hours. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. F and G, Upon treatment with increasing doses of BAY 2666605 for 24 hours, HEL (F) and U937 (G) cells were stained with annexin V/propidium iodide and subjected to flow cytometry. The percentage of apoptotic cells for HEL (F) and U937 (G) cells treated with increasing BAY 2666605 concentrations is shown as mean ± SEM of three independent experiments. Statistical significance was determined using a one-way ANOVA, followed by a Dunnett multiple comparisons test to adjust for multiple comparisons for each dose vs. control; **, P ≤ 0.01; ****, P ≤ 0.0001. H and I, Co-immunoprecipitation analysis of SLFN12 interaction with (H) PDE3A in HEL cells or (I) PDE3B in U937 cells. HEL and U937 cells were treated with low (1 μmol/L) or high (10 μmol/L) concentrations of BAY 2666605 for 24 hours. DMSO was used as vehicle control. Cell lysates were used for immunoprecipitation with anti-PDE3A and anti-PDE3B antibodies or control rabbit immunoglobulin, as indicated. H, After probing for PDE3A, the membrane was stripped and reprobed with antibody for PDE3B. BAY, BAY 2666605; Cl casp-3, cleaved caspase-3; IP, immunoprecipitation; RIgG, rabbit immunoglobulin; WCL, whole-cell lysate.

Figure S1. Dot plot of HEL and U937 cells undergoing apoptosis.

Effects of velcrin treatment on SLFN12 expression in vivo. A, Schematic representation of the treatment regimen used for the xenograft AML mouse model using BAY 2666605 or vehicle control. B, Tumor growth curves of nude (NU/NU) mice treated and challenged with HEL cells. Each point represents the mean tumor volume ± SEM per group for vehicle control (5% ethanol with 95% polyethylene glycol-400)– and BAY 2666605–treated mice (n = 5 for control; n = 4 for treated). Pink bar indicates days in which mice were treated with BAY 2666605 or vehicle control. Tumor growth from the start of treatment (day 14) until the end of treatment (day 18) was analyzed using a mixed-effects linear regression model. Tumor volume was log-transformed to satisfy the normality assumption. Treatment group, time, and their interaction were included as fixed effects and mice as a random effect. The interaction was statistically significant (P < 0.001). The P value comparing treatments at day 18 is reported (*, P = 0.011). C, Tumor lysates from the experiment shown in panel (B) were subjected to SDS-PAGE and probed for SLFN12 protein expression. D, Tumor samples from the same experiment (n = 4 per group) were subjected to RNA-seq. Volcano plot shows the differentially up- or downregulated genes between the velcrin treatment group and the vehicle control group. Genes deemed statistically significant are highlighted in red. Data are from four biological replicates per group (see also Supplementary Fig. S2). E and F, Gene ontology (GO) analysis of differentially expressed genes that were (E) downregulated or (F) upregulated in mouse tumors treated with BAY 2666605 compared with vehicle control–treated mice. Relevant pathways associated with differentially expressed genes are underlined in red. BAY, BAY 2666605; SQ, subcutaneous. [A, Created in BioRender. Guillen Magana, J. (2025) https://BioRender.com/aqz79jx.]

Antileukemic activity of BAY 2666605 in vivo. A, Schematic representation of the experimental approach for the treatment regimen used for the xenograft AML mouse model. B, Tumor volumes (means ± SEM) are shown for mice treated with either vehicle control (5% ethanol with 95% polyethylene glycol-400) or BAY 2666605 (5 mg/kg; n = 10 per group). Pink bars indicate treatment window. Tumor growth from the start of treatment (day 13) until the end of treatment (day 24) was analyzed using a mixed-effects linear regression model. Tumor volume was log-transformed to satisfy the normality assumption. Treatment group, time, and their interaction were included as fixed effects and mice as a random effect. The interaction was statistically significant (P < 0.0001). The P values comparing treatments at each time point were adjusted for multiple comparisons using the Holm–Šídák method; *, P ≤ 0.05. Arrowheads indicate when mice were euthanized because of meeting study endpoints after tumor volume measurement. C, Kaplan–Meier survival analysis for mice treated with BAY 2666605 vs. vehicle control. Mice were compassionately euthanized once tumor volume was ≥2,000 mm3 or weight loss was greater than 20%; ***, P = 0.0004 using a Mantel–Cox (log-rank) test. D, Table summarizing estimated median survival (in days) of mice from each treatment group. BAY, BAY 2666605; SQ, subcutaneous. [A, Created in BioRender. Guillen Magana, J. (2025) https://BioRender.com/yw4tt0f.]

We examined the role of SLFN12, a member of the Schlafen (SLFN) family of interferon-regulated genes and proteins in leukemogenesis, and its potential as a therapeutic target in acute myeloid leukemia (AML). We explored the effects of velcrins, a class of small molecules able to modulate SLFN12 biological activity, on AML cells. Velcrin treatment of AML cells stabilized SLFN12 and promoted SLFN12 complex formation with phosphodiesterase 3A or phosphodiesterase 3B. Such effects were associated with growth-inhibitory and proapoptotic responses, as well as potent suppressive effects on leukemic cell growth. In addition, velcrin treatment suppressed clonogenic capacity of primitive leukemic progenitors and significantly extended survival in a mouse AML xenograft model. Taken together, these findings establish an important role of SLFN12 in leukemogenesis and raise the potential for the use of velcrins as a therapeutic strategy for AML.Significance:Our studies identify SLFN12 as a potential target in AML with important clinical–translational implications.

Antileukemic effects of the velcrin–Aza combination. A and B, Cell viability of HEL (A) or U937 (B) cells treated with DMSO (vehicle control) or with increasing concentrations of BAY 2666605 (BAY), Aza, or the combination of BAY 2666605 with Aza as measured by WST-1 cell viability assay. Data are presented as the means ± SEM of three independent experiments, each conducted in triplicate, and are expressed as percent cell viability relative to vehicle control (DMSO)–treated cells. IC50 values for the indicated cell lines were calculated by fitting a nonlinear regression analysis using the “log (inhibitor) vs. response - three parameters” function in GraphPad Prism software (v.10). CI was calculated using Compusyn software, in which CI < 1 is synergistic, CI > 1 is antagonistic, and CI = 1 is additive. C and D, Clonogenic capability of (C) HEL or (D) U937 cells plated in a methylcellulose culture assay system in the presence of BAY 2666605, Aza, or the combination of BAY 2666605 with Aza, relative to vehicle control (DMSO; indicated by the dotted line) at the indicated concentrations. The percent of leukemic colony formation (CFU-L) is shown relative to vehicle control (DMSO)–treated cells and represents mean ± SEM of four independent experiments. Each dot represents an independent experiment. Statistical significance was determined using a one-way ANOVA, followed by a Tukey multiple comparisons test to correct for pairwise comparisons; ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. BAY, BAY 2666605.

Effects of velcrin treatment on leukemic progenitor colony formation. A and B, Clonogenic capability of HEL (A) or U937 (B) cells following treatment with increasing concentrations of BAY 2666605, as noted, for 1 week. Leukemic colony formation (CFU-L) was calculated as percent colony formation relative to vehicle control (DMSO)–treated cells (indicated by the dotted line) and means ± SEM of (A) three or (B) five independent experiments are presented. Each dot represents an independent experiment. At each concentration, statistical significance was determined using a one-sample Student t test compared with 100%; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. C, Effects on primary AML-derived leukemic progenitor colony formation. Cells were plated in a methylcellulose culture assay system and treated the same day with BAY 2666605 at the noted concentrations. Leukemic colony formation (CFU-L) was calculated as percent colony formation of BAY 2666605–treated patient samples (2 weeks) relative to vehicle control (DMSO)–treated cells (indicated by the dotted line). Each dot represents an independent experiment. Data are shown as means ± SEM of five independent experiments, as indicated, using primary cells from five different patients with AML. At each concentration, statistical significance was determined using a one-sample Student t test compared with 100%; ***, P ≤ 0.001; ****, P ≤ 0.0001. BAY, BAY 2666605.

SLFN12 expression and velcrin sensitivity of leukemic lines. A, Box plots of SLFN12 mRNA expression in various cancer types analyzed from the TCGA Pan-Cancer dataset using the University of California Santa Cruz Xena Browser portal. B, Immunoblotting analysis of PDE3B, PDE3A, and SLFN12 in established leukemia cell lines (HEL, K-562, KG-1, SET-2, U937, OCI-AML-5, and MV-4-11). C, Dose–response curves for cell viability of the indicated leukemia cell lines upon treatment with increasing concentrations of BAY 2666605 for 4 days, assessed by WST-1 assays. For each cell line, data represent means ± SEM of three independent experiments, each done in triplicate. D, IC50 values for the indicated lines were calculated by fitting a nonlinear regression analysis using the “log (inhibitor) vs. response - three parameters” function in GraphPad Prism software (v.10). E,SLFN12 mRNA expression of HEL cells was assessed by qRT-PCR normalized to GAPDH, using a non-targeting siRNA as a control. Data are expressed as the percentage of SLFN12 mRNA expression over control (%) and represent means ± SEM of three independent experiments, each conducted in triplicate. Statistical analysis was performed using a one-sample Student t test; **, P ≤ 0.01. F, Dose–response curve of HEL cells 4 days following transfection with control or SLFN12-targeting siRNA and treatment with increasing concentrations of BAY 2666605, as measured by WST-1. Data represent cell viability normalized to siRNA control and displayed as means ± SEM of three independent experiments, each conducted in triplicate. IC50 values for the indicated lines were calculated by fitting a nonlinear regression analysis using the “log (inhibitor) vs. response - three parameters” function in GraphPad Prism software (v.10). G, Clonogenic assay of HEL cells transfected with control or SLFN12-targeting siRNA and treated with the indicated concentrations of BAY 2666605 for 7 days. Leukemic colony formation (CFU-L) was calculated as percent colony formation relative to vehicle control (DMSO-treated cells) and means ± SEM of three independent experiments are presented. Each dot represents an independent experiment. Statistical significance was determined using an ordinary two-way ANOVA, followed by a Tukey multiple comparisons test; ns, not significant, *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. ACC, adrenocortical carcinoma; BAY, BAY 2666605, BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, PANCREATIC adenocarcinoma; PCPG, pheocromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Figure S3. Stable body weight and tumor volume post-BAY treatment in vivo.