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[Preprint]. 2024 May 24:2024.01.22.576669.
doi: 10.1101/2024.01.22.576669.

Targeting the mSWI/SNF Complex in POU2F-POU2AF Transcription Factor-Driven Malignancies

Tongchen He  1   2   3   4 , Lanbo Xiao  1   2   4 , Yuanyuan Qiao  1   2   5 , Olaf Klingbeil  6 , Eleanor Young  1   2 , Xiaoli S Wu  6 , Rahul Mannan  1   2 , Somnath Mahapatra  1   2 , Sanjana Eyunni  1   2 , Jean Ching-Yi Tien  1   2 , Xiaoju Wang  1   2 , Yang Zheng  1   2 , NamHoon Kim  1 , Heng Zheng  1   2 , Siyu Hou  7 , Fengyun Su  1   2 , Stephanie J Miner  1   2 , Rohit Mehra  1   2 , Xuhong Cao  1   2   8 , Chandrasekhar Abbineni  9 , Susanta Samajdar  9 , Murali Ramachandra  9 , Abhijit Parolia  1   2   5 , Christopher R Vakoc  6 , Arul M Chinnaiyan  1   2   5   8   10   11
Affiliations

Targeting the mSWI/SNF Complex in POU2F-POU2AF Transcription Factor-Driven Malignancies

Tongchen He et al. bioRxiv. .

Update in

  • Targeting the mSWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies.
    He T, Xiao L, Qiao Y, Klingbeil O, Young E, Wu XS, Mannan R, Mahapatra S, Redin E, Cho H, Bao Y, Kandarpa M, Ching-Yi Tien J, Wang X, Eyunni S, Zheng Y, Kim N, Zheng H, Hou S, Su F, Miner SJ, Mehra R, Cao X, Abbineni C, Samajdar S, Ramachandra M, Dhanasekaran SM, Talpaz M, Parolia A, Rudin CM, Vakoc CR, Chinnaiyan AM. He T, et al. Cancer Cell. 2024 Aug 12;42(8):1336-1351.e9. doi: 10.1016/j.ccell.2024.06.006. Epub 2024 Jul 18. Cancer Cell. 2024. PMID: 39029462 Free PMC article.

Abstract

The POU2F3-POU2AF2/3 (OCA-T1/2) transcription factor complex is the master regulator of the tuft cell lineage and tuft cell-like small cell lung cancer (SCLC). Here, we found that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex. SCLC-P cell lines were sensitive to nanomolar levels of a mSWI/SNF ATPase proteolysis targeting chimera (PROTAC) degrader when compared to other molecular subtypes of SCLC. POU2F3 and its cofactors were found to interact with components of the mSWI/SNF complex. The POU2F3 transcription factor complex was evicted from chromatin upon mSWI/SNF ATPase degradation, leading to attenuation of downstream oncogenic signaling in SCLC-P cells. A novel, orally bioavailable mSWI/SNF ATPase PROTAC degrader, AU-24118, demonstrated preferential efficacy in the SCLC-P relative to the SCLC-A subtype and significantly decreased tumor growth in preclinical models. AU-24118 did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F1/2 cofactor, POU2AF1 (OCA-B), were also remarkably sensitive to mSWI/SNF ATPase degradation. Mechanistically, mSWI/SNF ATPase degrader treatment in multiple myeloma cells compacted chromatin, dislodged POU2AF1 and IRF4, and decreased IRF4 signaling. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 enhanced survival compared to pomalidomide, an approved treatment for multiple myeloma. Taken together, our studies suggest that POU2F-POU2AF-driven malignancies have an intrinsic dependence on the mSWI/SNF complex, representing a therapeutic vulnerability.

Keywords: IRF4; POU2AF1/2/3; POU2F3; SMARCA2/4; mSWI/SNF complex; multiple myeloma; proteolysis targeting chimera (PROTAC); small cell lung cancer (SCLC).

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Figures

Figure 1.
Figure 1.. Dependence of SCLC-P cells on the mSWI/SNF complex.
(A) A schematic representation of the dual-sgRNA, domain-focused CRISPR screening designed to identify druggable epigenetic targets selective for SCLC subtypes. (B) Beta scores pertaining to all CRISPR screen targeted genes across both SCLC-P and SCLC-A cell lines (n = 5308). (C) Beta scores highlighting epigenetic regulators in SCLC-P and SCLC-A cell lines (n = 3292). (D) Percentage of different epigenetic complexes in SCLC-P and SCLC-A cell lines (top 10% for each). PRC1, polycomb repressive complex 1; PRC2, polycomb repressive complex 2; HDAC, histone deacetylase; TET, ten-eleven translocation family proteins. (E) Immunoblot analysis of indicated proteins in SCLC-P and SCLC-A cells post-treatment with varying time points or concentrations of AU-15330. Vinculin serves as the control for protein loading in all immunoblots. (F) Compilation of the IC50 values for AU-15330 in SCLC cell lines representing four molecular subtypes.
Figure 2.
Figure 2.. The POU2F3 transcription factor complex is evicted from chromatin in SCLC-P cells upon mSWI/SNF ATPase degradation.
(A) Chromatin compaction induced by mSWI/SNF ATPase degradation. Visualization of ATAC-seq read-density in NCI-H526 (SCLC-P) and NCI-H69 (SCLC-A) cells post-treatment for 4 hrs with either vehicle or 1 μM AU-15330. (B) Analysis of fold change and significance level for HOMER motifs that are enriched within sites dependent and independent of the mSWI/SNF complex in NCI-H526 and NCI-H69 cells. (C) ChIP–seq read-density heatmaps representing POU2F3 (green), HA-POU2F3 (red), and HA-POU2AF2 (blue) at AU-15330-compacted genomic sites in NCI-H526 cells following treatment with DMSO or AU-15330. (D) Volcano plot detailing proteins that interact with POU2AF2, as identified by POU2AF2 RIME analysis in NCI-H526 cells. mSWI/SNF components highlighted in orange. (E) Expression levels of POU2F3, POU2AF2/3, and PTGS1 as assessed by QPCR in the indicated cell lines after being treated with vehicle or AU-15330. (F) Volcano plot visualizing the overall transcriptomic alterations as assessed by RNA-seq in NCI-H526 and NCI-H1048 cells post-treatment with vehicle or AU-15330. Canonical POU2F3 target genes are highlighted in blue. (G) GSEA plots illustrating genes regulated by POU2F3 and its coactivators POU2AF2 and POU2AF3. The plots employ a gene signature ranked by fold change in AU-15330-treated NCI-H526 and NCI-1048 cells. DEG, differentially expressed gene. (H) Combined ATAC-seq and ChIP-seq tracks for AVIL, PTGS1, and ASCL2 in NCI-H526 with and without AU-15330 treatment.
Figure 3.
Figure 3.. Selective inhibition of SCLC-P xenograft tumor models employing an orally bioavailable mSWI/SNF ATPase degrader.
(A) Overview of the AU-24118 efficacy study conducted using SCLC xenograft models. (B) Analysis of tumor volume in indicated SCLC xenograft models upon treatment with AU-24118, measured bi-weekly using calipers (analyzed with a two-way ANOVA). (C) Immunoblot illustrating levels of the indicated proteins in SCLC-P and SCLC-A xenografts after 5 days of AU-24118 administration. Vinculin is utilized as the loading control across immunoblots. CDX, cell line-derived xenograft. (D) Representative H&E staining with corresponding IHC analyses for SMARCA4, POU2F3, and DCLK1 after 5 days of treatment with AU-24118 in NCI-H526 xenografts (scale=50μm). The inset scale=20μm. (E) (Left) Representative DAPI and TUNEL staining from xenografts from indicated cell lines after 5 days of AU-24118 treatment (scale=100μm). (Right) Quantitative evaluation of TUNEL staining of respective SCLC xenografts for 5 days. T-tests were used to calculate the significance. P value < 0.05 in the top panel. (F) Representative H&E staining of murine lung and small intestine with corresponding tuft cell marker DCLK1 IHC after in vivo administration of AU-24118 at study endpoint (scale=50μm). Magnified views of intestinal enterocytes and lung alveolar epithelium in H&E and corresponding DCLK1 IHC shown in insets (scale=20μm). (G) DCLK1 cell positivity in lung and small intestine for endpoint evaluation. AU-24118 (15mg/kg) dosed. Ns, not significant (t-tests).
Figure 4.
Figure 4.. POU2AF1-driven B cell malignancies are dependent on the mSWI/SNF complex.
(A) Scatter plot depicting gene dependency difference of all plasma cell myeloma versus other cancer types based on DepMap. The red circles indicate the top 5 essential genes among others. (B) Representative hematological cancer cell lines showing dose-response curves of AU-15330 at varying concentrations for five days. Sensitive cell lines are in red while relatively resistant cell lines in blue. (C) ATAC-seq read-density heatmaps from MM1.S cells treated with DMSO or 1 μM AU-15330 for 4 hours (n = 2 biological replicates). (D) Analysis of fold change and significance level for HOMER motifs that are enriched within sites dependent and independent of the mSWI/SNF complex after 4 hrs AU-15330 treatment in MM1.S cells (left panels) and NCI-H929 cells (right panels). (E) ChIP-seq read-density heat maps for POU2AF1 and IRF4 at the AU-15330-compacted genomic sites in MM1.S cells after treatment with DMSO or AU-15330 (1μM) for 6 hrs. (F) Volcano plot detailing proteins that interact with POU2AF1, as identified by POU2AF1 RIME analysis in MM1.S cells. mSWI/SNF components highlighted in orange. (G) GSEA plots illustrating genes regulated by IRF4. The plots use a gene signature ranked by fold change from AU-15330 treated NCI-H929 (top) and MM1.S (bottom) cells. (H) (Top) Analysis of tumor volumes in the MM1.S xenograft model upon treatment with AU-24118 and pomalidomide, measured bi-weekly using calipers (analyzed with a two-way ANOVA). (Bottom) Waterfall plot of tumor volumes at endpoint. (I) Overview of the MM1.S multiple myeloma disseminated xenograft model efficacy study. (J) Bioluminescence of images of MM1.S disseminated xenograft model after different treatments. The mice were monitored once per week. The signal intensity of bioluminescence represented the tumor burden (x108 photons/sec/cm2/steradian). Pomalidomide (10mg/kg) and AU-24118 (15mg/kg) dosed. (K) Kaplan-Meier survival curve of MM1.S disseminated xenograft model after pomalidomide (10mg/kg) and AU-24118 (15mg/kg) treatment. (L) Representative DAPI and TUNEL staining from the MM1.S disseminated xenograft model and quantitative evaluation from TUNEL staining for pomalidomide (10mg/kg) and AU-24118 (15mg/kg) treatment for 12 days. (M) Representative H&E and CD38 IHC staining of spinal vertebral marrow after in vivo administration of pomalidomide (10mg/kg) and AU-24118 (15mg/kg) for 12 days.
Figure 5.
Figure 5.. Schematic showing the mechanism of action of mSWI/SNF ATPase degraders in POU2F3-dependent SCLC and POU2AF1-dependent B cell malignancies.
The mSWI/SNF complex remodels nucleosomes at enhancer sites, allowing physical access to transcription factors (TFs) - POU2F3 in SCLC-P and IRF4 in multiple myeloma. Activity of the TFs is regulated by requisite coactivators (POU2AFs). This leads to recruitment of the transcriptional machinery at promoters, driving transcription of POU2F3 or IRF target genes. SMARCA2/4 ATPases are degraded with PROTAC treatment, resulting in loss of mSWI/SNF complex activity and eviction of TFs/coactivators and transcriptional machinery. This leads to diminished transcription of POU2F3 and IRF4 target genes and decreased oncogenic signaling from these pathways.
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