EVI1 (Ecotropic Viral Integration Site 1) is a zinc finger transcriptional regulator encoded from the MECOM (MDS1 and EVI1 Complex) locus at chromosome 3q26.2. Aberrant EVI1 overexpression is ...observed in approximately 10% of de novo AML, of which approximately half are due to chromosomal rearrangement involving either inv3(q21;q26.2) or t(3;3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is relocated and drives EVI1 expression. In addition to the resulting haploinsufficiency of GATA2, monosomy 7 and activating mutations in the RAS pathway and SF3B1 commonly co-occur in 3q26.2-rearranged (r) AML. AML with EVI1 overexpression generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Among the targets of EVI1 are the ERG, MYC, KIT, Bcl-xL and MPL genes. GATA2 repression, coupled with increased EVI1 activity, promotes AML progression and an aggressive phenotype in 3q26.2-rearranged AML. Therefore, there is a glaring need to develop and test novel targeted therapies with improved efficacy in repressing EVI1 and improving survival of AML with EVI1 overexpression. To identify druggable vulnerabilities in 3q26.2-r AML, we conducted an unbiased high-throughput drug screen, utilizing a library of 2480 oncology-focused, mechanistically annotated drugs (referred as NCATS Mechanism Interrogation Plates or-MIPE 5.0) (Cancer Cell. 2021; 39:566-579), against seven AML cell lines, i.e., four with 3q26.2-r: UCSD-AML1, HNT-34, AML191 and AML194, and three non-3q26.2-r AML: SET-2, MV4-11 and OCI-AML3. The library exploits mechanistic redundancy by including multiple inhibitors against well-validated oncology targets, while simultaneously encompassing mechanistic diversity, altogether targeting more than 850 distinct mechanisms of action. We exploited this redundancy to identify druggable, target-level dependencies in 3q26.2-r AML, both in terms of absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. This was consistent with previous reports that BET inhibitors (e.g., OTX015, mivebresib or ABBV-075 and JQ1) are effective against 3q26.2-r AML cell lines, patient-derived (PD) AML cells and PDX models. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, XIAP/cIAPs inhibitors birinapant (10-1000 nM) or SM-164 (30-1000 nM), chosen based on the MIPE screen outcomes, induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines. This was associated on Western analyses with decline in XIAP, c-IAP1/2, p-ERK1/2, MCL1 and Bcl-xL, but increased protein levels of cleaved caspase-3 and PARP in UCSD-AML1 and AML191 cells. Mivebresib (50-250 nM) or OTX015 (100-500 nM) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, XIAP, c-IAP1/2, CDK4/6 and Bcl-xL while increasing protein levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment with birinapant with mivebresib was synergistically lethal in UCSD-AML1 and AML191 cells, with Delta Synergy scores > 1.0 calculated by the ZIP method. Treatment with the dual mTOR/PIK3CA inhibitor NVP-BGT226 (1-30 nM) or navitoclax or Bcl-xL-specific BH3 mimetic A-1155463 also exerted lethality and synergistically induced apoptosis with mivebresib in AML cells with inv3/t(3;3). We previously reported that the TBL1/β-catenin antagonist tegavivint induced apoptosis in 3q26.2-r PD AML cells and PDX models. Co-treatment with birinapant and tegavivint also synergistically induced apoptosis in 3q26.2-r AML cells. Finally, in the xenograft model of AML194-Luc cells in NSG mice, monotherapy with birinapant (30 mg/kg/t.i.w, Intraperitoneal), vs vehicle control, significantly reduced the AML burden, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with birinapant and the BETi OTX015 (30 mg/kg/day, by oral gavage) was more effective in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of IAP protein inhibition against the cellular models of AML with inv3/t(3;3) with EVI1 overexpression, supporting the rationale to further evaluate in vivo efficacy of birinapant and/or BETi-based combinations against this AML sub-type.
Overexpression of immune-related genes is widely reported in acute myeloid leukemia (AML). Research from our lab and others has demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on ...innate immune signaling through IRAK4 (reviewed in Bennett et al., Blood 2023). Moreover, AML LSPCs express a hypermorphic isoform of IRAK4 (IRAK4-L), due to U2AF1- and SF3B1-dependent mis-splicing. Building on this preclinical evidence, several IRAK4 inhibitors are now undergoing clinical trials in AML patients. In Phase-1 findings with a selective IRAK4 inhibitor (CA-4948; Curis Therapeutics), it was found that MDS and AML patients with splicing factor mutations responded best to monotherapy IRAK4 inhibition, although the overall response rate with monotherapy was modest. These findings suggest that IRAK4 is a relevant target in myeloid malignancies, but the identification of synthetic lethal dependencies may be crucial in revealing effective combination therapies. In this study, we sought to identify synthetic lethal interactions upon IRAK4 inhibition in AML. For this, we first generated isogenic AML cells that are proficient (WT THP1) or deficient for IRAK4 (IRAK4 KO THP1) using site-directed mutagenesis with CRISPR-Cas9. Deletion of IRAK4 resulted in a modest (~50%) reduction in leukemic colony formation and negligible differences in growth potential. We used the isogenic WT and IRAK4 KO AML cells to screen for synthetic lethal interactions by performing a 2400+ drug in vitro screen. Hits were prioritized for potency in IRAK4 KO cells vs WT AML cells, degree of fitness, and overall effect. Among these hits, the Cereblon E3 ligase modulator (CELMoD) CC-885 emerged as the top target. Independent validation confirmed that CC-885 significantly decreased proliferation and viability of isogenic IRAK4 KO MDS (MDSL) and AML (THP1) cells by approximately 3-4-fold compared to WT MDS or AML cells. Colony formation in methylcellulose after treatment with CC-885 was also significantly reduced in IRAK4 KO compared to WT MDS and AML cells. Based on Caspase-3 activation and AnnexinV staining, CC-885 induced significantly more apoptosis of IRAK4 KO cells as compared to WT AML cells. We extended our synthetic lethal analysis to a clinical-stage IRAK4 inhibitor (CA-4948). MDS and AML cells were treated with CA-4948 concurrently with CC-885 or pretreated for 7 days prior to treatment with CC-885. Unexpectedly, we found that only prior exposure of the AML cells to the IRAK4 inhibitors resulted in sensitivity to CC-885. These findings indicate that IRAK4 inhibition reprograms AML cells so that they acquire a synthetic lethal interaction with CC-885. CC-885 is known to target multiple substrates for degradation by the ubiquitin proteasome, including IKZF1, IKZF3, CK1a, and GSPT1. We compared the effect of CC-885 to CC-90009, a CELMoD with GSPT1 specific activity in the isogenic IRAK4 KO and WT AML cell lines. CC-90009 did not result in complete cell death up to concentrations of 10mM in both WT and IRAK4 KO, suggesting that the selective sensitivity of IRAK4 KO AML cells to CC-885 is not due to inhibition of GSPT1. In support of these interpretations, IRAK4 deficient AML cells exhibit increased protein expression of IKZF2 and IKZF3, but not GSPT1 or CK1a. Given the unique responses of IRAK4 KO cells to CELMoDs, we posited that IRAK4 inhibition alters the neosubstrate profile of CELMoDs in AML cells. Total proteome characterization of CC-885 treated WT and IRAK4 KO THP1 cells by mass spectrometry identified a significant decrease in 90 protein substrates in IRAK4 KO cells treated with CC-885 as compared to WT cells treated with CC-885. These findings suggest that IRAK4 inhibition alters the pool of neosubstrates in AML cells for certain CELMoDs. Overall, our study demonstrates that IRAK4 is a therapeutic target in AML, but that combination therapies, such as with certain CELMoDs, will be necessary to achieve better clinical responses.
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive and largely incurable hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). Using RNAi screening, we ...identified the E-box transcription factor TCF4 as a master regulator of the BPDCN oncogenic program. TCF4 served as a faithful diagnostic marker of BPDCN, and its downregulation caused the loss of the BPDCN-specific gene expression program and apoptosis. High-throughput drug screening revealed that bromodomain and extra-terminal domain inhibitors (BETis) induced BPDCN apoptosis, which was attributable to disruption of a BPDCN-specific transcriptional network controlled by TCF4-dependent super-enhancers. BETis retarded the growth of BPDCN xenografts, supporting their clinical evaluation in this recalcitrant malignancy.
•The E-box transcription factor TCF4 is a lineage-survival oncogene in BPDCN•BET inhibitors are highly toxic to BPDCNs, both in vitro and in vivo•A druggable TCF4/BRD4 transcriptional network sustains malignancy in BPDCN•BET inhibitors should be explored for the clinical management of BPDCN
Ceribelli et al. use a combination of RNAi and small-molecule screening to identify TCF4 as a crucial transcriptional regulator required for maintenance of blastic plasmacytoid dendritic cell neoplasm (BPDCN) and show that bromodomain inhibitors are potential therapeutics for BPDCN, through targeting of TCF4.
Antisense transcription has been shown to be one of the hierarchies that control gene expression in eukaryotes. Recently,
we have documented that the mouse Kcnq1 imprinting control region (ICR) ...harbors bidirectional silencing property, and this feature is linked to an antisense RNA,
Kcnq1ot1 . In this investigation, using genomic footprinting, we have identified three NF-Y transcription factor binding sites appearing
in a methylation-sensitive manner in the Kcnq1ot1 promoter. By employing a dominant negative mutant to the NF-Y transcription factor, we have shown that the NF-Y transcription
factor positively regulates antisense transcription. Selective mutation of the conserved nucleotides in the NF-Y binding sites
resulted in the loss of antisense transcription. The loss of antisense transcription from the Kcnq1ot1 promoter coincides with an enrichment in the levels of deacetylation and methylation at the lysine 9 residue of histone H3
and DNA methylation at the CpG residues, implying a crucial role for the NF-Y transcription factor in organizing the parent
of origin-specific chromatin conformation in the Kcnq1 ICR. Parallel to the loss of antisense transcription, the loss of silencing of the flanking reporter genes was observed,
suggesting that NF-Y-mediated Kcnq1ot1 transcription is critical in the bidirectional silencing process of the Kcnq1 ICR. These data highlight the NF-Y transcription factor as a crucial regulator of antisense promoter-mediated bidirectional
silencing and the parent of origin-specific epigenetic marks at the Kcnq1 ICR. More importantly, for the first time, we document that NF-Y is involved in maintaining the antisense promoter activity
against strong silencing conditions.
Schlafen 11 (SLFN11) is an increasingly prominent predictive biomarker and a molecular sensor for a wide range of clinical drugs: topoisomerases, PARP and replication inhibitors, and platinum ...derivatives. To expand the spectrum of drugs and pathways targeting SLFN11, we ran a high-throughput screen with 1,978 mechanistically annotated, oncology-focused compounds in two isogenic pairs of SLFN11-proficient and -deficient cells (CCRF-CEM and K562). We identified 29 hit compounds that selectively kill SLFN11-proficient cells, including not only previously known DNA-targeting agents, but also the neddylation inhibitor pevonedistat (MLN-4924) and the DNA polymerase α inhibitor AHPN/CD437, which both induced SLFN11 chromatin recruitment. By inactivating cullin-ring E3 ligases, pevonedistat acts as an anticancer agent partly by inducing unscheduled re-replication through supraphysiologic accumulation of CDT1, an essential factor for replication initiation. Unlike the known DNA-targeting agents and AHPN/CD437 that recruit SLFN11 onto chromatin in 4 hours, pevonedistat recruited SLFN11 at late time points (24 hours). While pevonedistat induced unscheduled re-replication in SLFN11-deficient cells after 24 hours, the re-replication was largely blocked in SLFN11-proficient cells. The positive correlation between sensitivity to pevonedistat and SLFN11 expression was also observed in non-isogenic cancer cells in three independent cancer cell databases (NCI-60, CTRP: Cancer Therapeutics Response Portal and GDSC: Genomic of Drug Sensitivity in Cancer). The present study reveals that SLFN11 not only detects stressed replication but also inhibits unscheduled re-replication induced by pevonedistat, thereby enhancing its anticancer efficacy. It also suggests SLFN11 as a potential predictive biomarker for pevonedistat in ongoing and future clinical trials.
Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating ...functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin. Deleting Arid1a in activated murine B cells disrupts IRF4-dependent transcriptional networks and blocks plasma cell differentiation. Targeting SWI/SNF activity leads to rapid loss of IRF4-target gene expression and quenches global amplification of oncogenic gene expression by MYC, resulting in profound toxicity to MM cells. Notably, MM patients with aggressive disease bear the signature of SWI/SNF activity, and SMARCA2/4 inhibitors remain effective in immunomodulatory drug (IMiD)-resistant MM cells. Moreover, combinations of SWI/SNF and MEK inhibitors demonstrate synergistic toxicity to MM cells, providing a promising strategy for relapsed/refractory disease.
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•ARID1A promotes IRF4 expression through the cBAF complex•ARID1A is required for plasma cell development and myeloma survival•SMARCA2/4 inhibition disrupts oncogenic transcription in multiple myeloma•SMARCA2/4 inhibition overcomes IMiD resistance and synergizes with MEK inhibitors
IRF4 orchestrates oncogenic transcription in multiple myeloma. Here, Bolomsky et al. employ a multi-omic examination of IRF4 in multiple myeloma to reveal that ARID1A and SWI/SNF activity are required for IRF4-dependent transcription. Inhibition of SWI/SNF blocks plasma cell development and is toxic to myeloma cells, including cells resistant to IMiDs.
Major advances have been made in the field of precision medicine for treating cancer. However, many open questions remain that need to be answered to realize the goal of matching every patient with ...cancer to the most efficacious therapy. To facilitate these efforts, we have developed CellMinerCDB: National Center for Advancing Translational Sciences (NCATS; https://discover.nci.nih.gov/rsconnect/cellminercdb_ncats/), which makes available activity information for 2,675 drugs and compounds, including multiple nononcology drugs and 1,866 drugs and compounds unique to the NCATS. CellMinerCDB: NCATS comprises 183 cancer cell lines, with 72 unique to NCATS, including some from previously understudied tissues of origin. Multiple forms of data from different institutes are integrated, including single and combination drug activity, DNA copy number, methylation and mutation, transcriptome, protein levels, histone acetylation and methylation, metabolites, CRISPR, and miscellaneous signatures. Curation of cell lines and drug names enables cross-database (CDB) analyses. Comparison of the datasets is made possible by the overlap between cell lines and drugs across databases. Multiple univariate and multivariate analysis tools are built-in, including linear regression and LASSO. Examples have been presented here for the clinical topoisomerase I (TOP1) inhibitors topotecan and irinotecan/SN-38. This web application provides both substantial new data and significant pharmacogenomic integration, allowing exploration of interrelationships.
CellMinerCDB: NCATS provides activity information for 2,675 drugs in 183 cancer cell lines and analysis tools to facilitate pharmacogenomic research and to identify determinants of response.
Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive ...B-cell lymphomas, including diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). GCs act by binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. The transcriptional response to GCs can vary significantly due to the variation in expression levels of GR cofactors and chromatin landscapes in different cell types. The mechanisms by which GCs kill malignant lymphoma cells are largely unknown, prompting us to search for new targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To identify genes that synergize or antagonize GC lethality in lymphomas, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone, the active metabolite of prednisone. Screens in cell line models of BL and DLBCL (both ABC and GCB subtypes) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor signaling pathway, which is required to sustain the viability of these malignant lymphoma cells. With combination of the cleavage under targets and release using nuclease (CUT&RUN) assay and RNA-seq, we identified GR binding to the LAPTM5 locus at its glucocorticoid response element and AP1 motifs upon GC treatment. GR binding also induced expression of LAPTM5, which negatively regulated BCR signaling by promoting the lysosomal degradation of the BCR. Conversely, GC induced binding of GR to the CSK locus, thereby repressing expression of the non-receptor tyrosine kinase CSK, which antagonizes BCR signaling by phosphorylating an inhibitory tyrosine residue present in all Src-family kinases (SFKs). However, in BCR-dependent aggressive lymphomas, inactivation of CSK paradoxically decreased proximal BCR signaling and induced cell death. By performing quantitative phosphoproteome and ubiquitinome with mass spectrometry, we demonstrated that treatment of lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi) initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-kB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs, which have been used empirically for decades to treat lymphomas. GCs restrain the most proximal steps in BCR signaling at the plasma membrane by, on one hand, decreasing BCR abundance, and on the other hand, by decreasing CSK expression, thereby reducing expression of the essential SFKs. Small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers.
Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids ...inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.
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•Dampening oncogenic BCR signaling is a key anti-lymphoma action of glucocorticoids•The glucocorticoid receptor directly transactivates genes promoting BCR degradation•The glucocorticoid receptor represses CSK, degrading BCR-proximal kinases•Glucocorticoids are mechanistically rational drugs in anti-lymphoma regimens
For more than 70 years, glucocorticoids have been known to have clinical activity against lymphoid but not myeloid malignancies. Choi et al. resolve this conundrum by demonstrating that glucocorticoids kill lymphomas by inhibiting BCR signaling. These mechanistic insights enable rational combination of glucocorticoids with other targeted agents in anti-lymphoma regimens.
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