Flow cytometry

Measurements were made on a Sony SH800 (RRID: SCR_018066), BD FACSCanto (RRID: SCR_018055), BD LSR II (RRID: SCR_002159), or BD FACSAria II (RRID: SCR_018934). Single-stained cells or compensation beads (Invitrogen, 01–3333–41) were used as compensation controls. Antibodies and filter sets for each instrument are listed in Supplementary Materials and Methods. All data were analyzed using FlowJo v10.4.1 (RRID: SCR_008520).

Colony forming assays

Cells were counted and resuspended in IMDM 10% heat-inactivated FBS 1% pen/strep (human) or DMEM 2% heat-inactivated FBS 1% pen/strep at a 1:50 dilution (murine). Cells were plated in triplicate at 10,000 (human AML), 500 (human CD34⁺), or 1,000 (murine MLL-AF9) viable cells/plate using human or mouse MethoCult methylcellulose media (StemCell Technologies, 04435 or 03434) with indicated concentrations of BRM014 or DMSO. After 10 to 14 days, plates were scored for colony counts and cells analyzed by flow cytometry where indicated.

Genome-wide analyses

Human primary leukemias and CD34⁺ HSPCs

Mice

Female 6-to-8-week-old C57BL/6J mice were purchased from the Baylor College of Medicine (BCM) Animal Core Facility. All animal experiments performed in this study were approved by the BCM Institutional Animal Care and Use Committee and BCM Veterinary Office.

Hematopoiesis mouse model

Syngeneic KMT2A-MLLT3 mouse model

Statistical analysis

Statistical tests were performed in R (3.6.1) as two-sided tests (RRID: SCR_001905). For multiple-comparison tests, P values were adjusted using the Benjamini–Hochberg FDR correction procedure. P values below the 64-bit double precision machine epsilon (2⁻⁵² = 2.22e−16) are reported as P < 2.2e−16. Independent biological replicates were chosen in advance and are indicated in figure legends. All N values indicate independent biological replicates, genome-wide studies used N = 2 per condition in agreement with recommendations of the ENCODE Consortium, flow cytometry data are presented as representative analyses from N ≥ 3 replicates, and single-cell experiments used N = 3 pooled animals per condition.

PU.1 and SWI/SNF activity converge on the BENC module

In hematopoietic cells, MYC expression is regulated by the BENC module (13, 15). In AML cells, BRM014 treatment induced loss of DNA accessibility and CRC factor binding at BENC compared with DMSO (Fig. 2A; Supplementary Figs. S3A–S3B). Although PU.1 was retained at some BENC sites, binding partners such as RUNX1 showed strong reductions at SWI/SNF-bound sites across the region (Fig. 2A). Inhibition of PU.1 resulted in reduced SMARCA4 recruitment to PU.1 sites within BENC (Supplementary Fig. S3C), consistent with the PU.1-directed SWI/SNF recruitment described above.

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Figure 2.

Collaborative regulation of the BENC module underlies convergent differentiation induced by SWI/SNF or PU.1 inhibition in AML. A, Overlay of SMARCA4, PU.1, RUNX1, LMO2, and MEIS1 occupancy with ATAC-seq and RNA-seq data at MYC and BENC in THP-1 cells. Other cell lines are presented in Supplementary Figs. S3A and S3B. B and C, Expression of MYC and PU.1 in AML cells lines treated with BRM014 or DB2313 by RNA-seq (THP-1) or RT-qPCR (MV-4–11 and MOLM-13) at 72 hours (N = 3; B) and Western blot analysis at 24 hours of treatment (C). D, Genome-wide correlation of BRM014- and DB2313-induced transcriptional changes in THP-1 cells based on RNA-seq. E, Gene sets downregulated upon BRM014 and DB2313 treatment. Normalized enrichment scores (NES) are indicated. F, Cell surface expression of myeloid differentiation markers CD14 and CD87 in cells treated with DMSO, BRM014, or DB2313. G, Expression of CD44 in cells treated with DMSO, BRM014, DB2313, or PMA measured by RNA-seq (THP-1) or RT-qPCR (MV-4–11 and MOLM-13) at 72 hours (N = 3). H, Expression of CD14 and CD44 in cells treated with DMSO, BRM014, DB2313, or PMA measured by flow cytometry. Error bars, mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Treatment with either BRM014 or DB2313 caused loss of MYC RNA expression in AML cell lines (Fig. 2B). MYC protein levels were similarly reduced in MV-4–11 and MOLM-13 cells within 24 hours, but required 72 hours for complete reduction in THP-1 cells treated with DB2313 (Fig. 2C; Supplementary Fig. S3D). Despite modest reduction of PU.1 levels in some cell lines consistent with its autoregulatory function (50), PU.1 remained expressed and was retained at its chromatin binding sites. However, loss of accessibility at PU.1 motifs (Fig. 1A) arose even in cells lacking strong PU.1 expression changes (Fig. 2C).

Transcriptional changes induced by BRM014 and DB2313 treatment were strongly correlated genome wide (Spearman R = 0.38 P < 2.2e-16; Fig. 2D), and included downregulation of MYC targets, genes repressed during myeloid development, and HSC-related genes (Fig. 2E). Both inhibitors induced leukemic differentiation, as confirmed by increased expression of the cell surface markers CD14 and CD87 (Fig. 2F). Interestingly, SWI/SNF and PU.1 inhibition also induced loss of the stemness marker (51) CD44, which is not a feature of phorbol 12-myristate 13-acetate (PMA)-induced differentiation of AML cells (Fig. 2G and ​andHH and Supplementary Fig. S3E), reflecting a unique signature of differentiation via PU.1 deregulation. BRM014 and DB2313 also exhibited largely additive effects (Supplementary Fig. S3F), consistent with convergence on the same pathway. Together, our findings reveal that PU.1 and SWI/SNF collaboratively regulate MYC expression in AML and that inhibition of either factor converges on myeloid differentiation.

SWI/SNF inhibition has off-tumor effects on PU.1-dependent hematopoietic cell types

Because SWI/SNF and PU.1 are both critical regulators of hematopoiesis, we sought to examine the effects of SWI/SNF inhibition on nontumor hematopoietic cells in vivo. We treated healthy C57BL/6 mice with daily oral gavage of 20 mg/kg BRM014 or vehicle control for 14 days (Fig. 3A), which was well tolerated and did not cause significant weight loss (P = 0.49, Supplementary Fig. S4A). After the 14-day treatment period, we observed decreased white blood cell (WBC) counts in the peripheral blood of BRM014-treated mice but no change in hemoglobin levels (Fig. 3B).

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Figure 3.

SWI/SNF inhibition induces reversible leukopenia in PU.1-dependent nontumor peripheral blood lineages. A, Dosing scheme for in vivo treatment of healthy mice. B, WBC and hemoglobin (HGB) levels in peripheral blood at day 14. N = 6, vehicle; N = 4, BRM014. C, Cell type–specific transcriptional markers were unchanged by treatment, enabling consistent classification. D, Composition of nongranulocyte immune cell types in the peripheral blood of BRM014- and DMSO-treated mice. E, Ranking of significantly reduced differential TF motif accessibility by immune population from mice treated with BRM014 versus vehicle control. F, UMAP embedding of individual cells by snATAC profile, with overlayed PU.1 motif accessibility. G, Quantification of accessible PU.1 motifs across specific cell types. H, Shifts of accessible DNA sites containing PU.1 motifs in B cells and monocytes upon BRM014 treatment. I, Total number of peripheral neutrophils and monocytes at day 14 and Ly6c expression in circulating monocytes. N = 3 per condition. J, Total number of T and B cells at day 14, with B-cell gating strategy. N = 6, vehicle; N = 4, BRM014. K, Total WBC, lymphocyte, and myeloid cell counts at day 14 and day 35 (3 weeks posttreatment) measured by CBC. Day 14, N = 6 (vehicle) and N = 4 (BRM014). Day 35, N = 3 (vehicle) and N = 2 (BRM014). Error bars, mean ± SEM; *, P < 0.05; **, P < 0.01; n.s., nonsignificant.

Single-nuclei multiome (sn-multiome; integrated snATAC and snRNA-seq) analysis of peripheral blood collected from BRM014- and vehicle-treated mice (N = 3 per condition) revealed cells in distinct clusters, which we identified as B cells, T cells, NK cells, and monocytes using established markers (Fig. 3C; Supplementary Fig. S4B). SWI/SNF inhibition caused a notable reduction in the proportion of peripheral B cells and monocytes (Fig. 3D), but gene expression changes were observed in all cell types (Supplementary Fig. S4C). PU.1 motifs were among the most significantly down-regulated TF motifs in B cells and monocytes, but not in NK or T cells (Fig. 3E). Moreover, reduced cell numbers corresponded to PU.1 motif utilization, with B cells and monocytes exhibiting the highest PU.1 motif accessibility, which was furthermore reduced by SWI/SNF inhibition (Fig. 3F and ​andG).G). Remarkably, more detailed analysis of sites with PU.1 motifs revealed DNA accessibility losses at distal sites but gains at promoters (Fig. 3H), similar to the effects observed in AML cells.

Flow cytometry (Fig. 3I and ​andJ)J) confirmed that compared with vehicle treatment, BRM014 induced no significant effect on neutrophils (P = 0.18) but reduced total monocytes 1.8-fold (P = 0.05), with specific loss of the Ly6C^high population, a marker for monocytes recently derived from BM (P = 3.7e−3, Fig. 3I; Supplementary Fig. S4D; refs. 52, 53). Within the lymphoid compartment, BRM014 treatment had no significant effect on peripheral T cells (P = 0.38) but caused a 5-fold depletion of B cells (P = 0.01, Fig. 3J; Supplementary Fig. S4E). Our findings reveal important off-tumor effects of SWI/SNF inhibition that arise through the same mechanisms responsible for therapeutic effects against AML.

To evaluate the reversibility of SWI/SNF inhibition's effects on hematopoiesis, we assessed CBCs from mice on the last day of treatment and 3 weeks after cessation of treatment. This comparison revealed that BRM014-induced effects on peripheral blood cells recover within 3 weeks of drug withdrawal (Fig. 3K), demonstrating that hematological and immune-related effects of transient SWI/SNF inhibition are reversible.

Effects of SWI/SNF inhibition on hematopoiesis arise within BM

To determine at which stage of hematopoietic development SWI/SNF sensitivity arises, we evaluated the effect of BRM014 on BM harvested from BRM014- and vehicle-treated mice at day 14 (N = 3 per condition). BM from BRM014-treated mice contained fewer total cells than vehicle control (P = 0.017, Fig. 4A). To assess the chromatin effects of BRM014 treatment, we performed sn-multiome on BM harvested at day 14, which revealed distinct clusters that we classified as B cells, erythroid cells, monocytes, T cells, plasmacytoid dendritic cells (pDC), and Kit⁺ hematopoietic stem and progenitor cells (HSPC) using standard cell-type-specific markers (Supplementary Fig. S5A). This study revealed the same loss of PU.1 motifs in B cells (P = 5.9e−17) and monocytes (P = 7.4e−3) in BM as observed in the peripheral blood, with no other cell types showing significant reductions (Fig. 4B; Supplementary Figs. S5A–S5C).

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Figure 4.

Transient SWI/SNF inhibition depletes committed hematopoietic cells in BM but preserves HSC function. A, Total number of cells isolated from BM of BRM014- and vehicle-treated mice at day 14. N = 3 per condition. B, Quantification of accessible PU.1 motifs across specific BM cell types. M, monocytes; B, B cells; Kit⁺, Kit⁺ progenitors; T, T cells; NK, NK cells. C, Cell type–specific transcriptional markers were unchanged by treatment, enabling consistent classification. UMAP embedding of individual cells pooled from all conditions. Resulting clusters are classified by immune cell type based on cell-specific expression profiles. D, Inferred total number of HSPC Kit⁺ and lineage-committed Kit^– cells determined by scRNA-seq. E, Flow cytometry quantification and histogram of lineage-committed (CD3, CD8, B220, GR1, TER119) and uncommitted CD45⁺ BM cells. F, Inferred total number of B cell and monocyte populations in BM determined by scRNA-seq. G, Total number of LSK (Lin^–Sca-1⁺Kit⁺) HSPCs in BM by flow cytometry. N = 3 per condition. H and I, Competitive HSCT workflow (H) and representative flow plots (I) of HSC engraftment and recapitulation of major hematopoietic lineages. N = 4, vehicle; N = 6, BRM014. Error bars, mean ± SEM. n.s., nonsignificant.

To provide increased resolution of expression changes within BM populations, we performed single-cell RNA-seq (scRNA-seq), which revealed similar clustering of cell identities but with increased granularity (Fig. 4C). Interestingly, scRNA-seq revealed reduced BM cell counts to be caused primarily by a loss of lineage-committed (Kit^–) cells (Fig. 4D). This finding was orthogonally confirmed by flow cytometry, which showed that SWI/SNF inhibition differentially affects BM cells that have become committed and lineage-restricted (P = 7.0e−3, Fig. 4E; Supplementary Figs. S5D–S5E). Further, reductions of B cells and monocytes varied between early proliferative cells and late nonproliferative cells across cell types (Fig. 4F), demonstrating that SWI/SNF inhibition has lineage-specific effects and is required at distinct developmental stages for each lineage (Supplementary Figs. S5F–S5G).

Importantly, BRM014 did not alter the number of LSK (Lin^–Sca-1⁺Kit⁺) HSPCs in BM (P = 0.57, Fig. 4G; Supplementary Fig. S5H). To assess the effects of SWI/SNF inhibition on HSC fitness and function, we performed a competitive HSC transplant of BM harvested from BRM014- and vehicle-treated mice (Fig. 4H). Excitingly, HSCs isolated from BRM014-treated mice retained the ability to engraft and to reconstitute all major hematopoietic lineages (Fig. 4I). We conclude that transient SWI/SNF inhibition does not permanently suppress HSC function.

SWI/SNF inhibition induces regression of AML in an immunocompetent in vivo model

To assess the efficacy of SWI/SNF inhibition against AML in vivo, we employed a syngeneic KMT2A-MLLT3 (“MLL-AF9”) leukemic model in C57BL/6 mice, which is an aggressive and immunocompetent AML model (54) with predictable latency. In vitro, leukemic cells displayed a dose-dependent inhibition of colony-forming capacity upon treatment with BRM014 (Supplementary Fig. S6A). We treated mice engrafted with GFP⁺ MLL-AF9 cells with BRM014 or vehicle control for 14 days (Fig. 5A). Compared with vehicle control, BRM014 strongly prevented leukemic expansion (P < 2.2e−16, two-way ANOVA, Fig. 5B and ​andC).C). Notably, we observed significant regression of disease burden (P = 8.3e−3), including one mouse in which the peripheral leukemic burden was reduced from 32% to 7% after 6 days of treatment (Supplementary Fig. S6B).

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Figure 5.

SWI/SNF inhibition has a significant therapeutic window against AML in an immunocompetent setting. A, Dosing scheme for in vivo treatment of MLL-AF9 leukemia with BRM014 or vehicle control. B and C, Leukemic burden of mice treated with BRM014 or vehicle control over the two-week treatment period (B) and quantification by flow cytometry (C). N = 11, vehicle; N = 10, BRM014. D, Spleens harvested after 14 days of treatment with BRM014 or vehicle control. E–G, Leukemic burden in the spleen (E), liver (F), and BM (G) of mice treated with BRM014 or vehicle control. N = 3 per condition. H–J, Effect of BRM014 on the number of committed (Lin⁺; H), uncommitted (Lin^–; I), and LSC-enriched Lin^–Sca-1⁻Kit⁺ stem/progenitor cells (J) within the leukemic population in BM. Cells in H, I, and J were gated on the basis of GFP⁺ cells. N = 3 per condition. K–M, Expression of stemness (CD44; K) and myeloid differentiation markers CD11B (L) and CD14 (M) on leukemic GFP⁺ cells in the BM of mice treated with BRM014 or vehicle control. N = 3 per condition. N, CBC neutrophil, monocyte, and lymphocyte counts and hemoglobin levels in BRM014-treated mice versus nonleukemic controls 3 days after completion of treatment course. N = 3, nonleukemic; N = 7, BRM014. O, Survival of MLL-AF9 mice treated with BRM014 or vehicle control. N = 6, untreated; N = 11, vehicle; N = 10, BRM014. Error bars, mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Treatment with BRM014 prevented leukemia-induced splenomegaly (P = 2.4e−4) and hepatomegaly (P = 5.5e−3, Fig. 5D; Supplementary Figs. S6C–S6D), with leukemic burden reduced 14.8-fold (P = 4.8e−6) in the spleen and 18.8-fold (P = 3.3e−5) in the liver (Fig. 5E and ​andF).F). Importantly, BRM014 also reduced leukemic burden in BM 7-fold compared with vehicle control (P = 1.2e−4, Fig. 5G). To understand the leukemic subpopulations affected by SWI/SNF inhibition, we assessed the effects on committed lineage-marker positive (Lin⁺) and immature (Lin^–) leukemic blasts (Fig. 5H and ​andI).I). BRM014 effectively reduced all leukemic subpopulations, resulting in a 5.3-fold reduction of Lin⁺ (P = 3.9e−4) and a 12-fold reduction of Lin^– leukemic BM cells (P = 1.9e−5). Notably, Lin^–Sca-1^–Kit⁺ leukemic cells, a population enriched for leukemic stem cells (55, 56), were reduced 20-fold (P = 1.8e−3, Fig. 5J).

To detect leukemic differentiation in vivo, we assessed the expression of CD44 and myeloid differentiation markers CD11B and CD14. Leukemic BM cells from mice treated with BRM014 for 14 days displayed decreased CD44 (P = 2e−3) and increased expression of CD11B (P = 0.05) and CD14 (P = 4.7e−5, Fig. 5K–M). Similar results were observed in the peripheral blood as early as day 9 (Supplementary Figs. S6E–S6G). Hence, our results confirm that leukemic differentiation occurs in both peripheral blood and BM in an immunocompetent context.

BRM014 was well tolerated and caused no significant weight loss (P > 0.05, two-way ANOVA; Supplementary Fig. S6H). CBCs 3 days after completion of therapy revealed vehicle-treated mice had high WBC counts, anemia, and thrombocytopenia, reflecting their leukemic state (Supplementary Figs. S6I–S6K). BRM014-treated mice had reduced WBC (P = 2.3e−4) and lymphocyte (P = 1.1e−5) counts compared with nonleukemic control mice (Fig. 5N, Supplementary Figs. S6I–S6K; Supplementary Dataset S4). Platelets, neutrophils, and monocytes were not significantly different (P > 0.05 in all cases). While hemoglobin was mildly reduced in BRM014-treated mice compared with normal controls (P = 6.1e−6), BRM014-treated mice were not anemic, with hemoglobin levels within a normal range. The modest inflation of monocytes in BRM014-treated mice was nonsignificant and caused by the identification of differentiated leukemic blasts as monocytes by CBC. However, flow cytometry of GFP-negative cells revealed significant reduction of nontumor myeloid cells in BRM014-treated compared with nonleukemic reference mice, confirming similar off-tumor effects in a leukemic setting (Supplementary Figs. S6L–S6M).

Treatment with BRM014 significantly prolonged survival (P = 1.9e−4, log-rank test), with a median survival of 44 days for BRM014-treated mice compared with 16 days for vehicle-treated mice (Fig. 5O). Overall, our results demonstrate that small-molecule inhibition of SWI/SNF ATPase activity significantly reduces disease burden and prolongs survival of AML.

The authors thank Zainab Jagani and Florencia Rago (Novartis) for helpful comments and feedback. This work was supported by grants to H.C. Hodges from Gabrielle's Angel Foundation, the V Foundation (V2018–003), the Mark Foundation for Cancer Research (20–024-ASP), and the Adrienne Helis Malvin Medical Research Foundation. H.C. Hodges is a CPRIT Scholar supported by grants from the NIH (R35GM137996 and R01CA272769) and the Cancer Prevention and Research Institute of Texas (CPRIT RR170036). C. Chambers was supported by NIH grant F31AI161906. H.D. Lacorazza was supported by NIH grant R01CA207086 and the ASH Bridge Award. This project was also supported by the UTMB Next-Generation Sequencing Core, the BCM Cytometry and Cell Sorting Core (CPRIT RP180672, NIH P30CA125123, and S10RR024574), the BCM Single Cell Genomics Core (NIH S10OD018033, S10OD023469, S10OD025240, and P30EY002520), the BCM Genomic and RNA Profiling Core (NIH S10OD023469), the Texas Children's Cancer Center tissue repository, and the MD Anderson Next-Generation Sequencing Core (CPRIT RP120348).

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