The CDK4/6-UCHL5-BRD4 axis confers resistance to BET inhibitors in MLL-rearranged leukemia cells by suppressing BRD4 protein degradation

2021 ◽  
Author(s):  
Keigo Amari ◽  
Satoru Sasagawa ◽  
Natsuki Imayoshi ◽  
Yuki Toda ◽  
Shigekuni Hosogi ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Leukemia Cells ◽  
Bet Inhibitors

scholarly journals Synthetic Lethality of Combinatory Inhibition of BET and CDK7 in BET(i) Resistant Leukemia Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1354-1354
Author(s):  
Lei Guo ◽  
Jia Li ◽  
Shaohai Fang ◽  
Minjung Lee ◽  
Hongxiang Zeng ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Drug Treatment ◽  
Synthetic Lethality ◽  
Single Agent ◽  
Factor H ◽  
Leukemia Cells ◽  
Super Enhancer ◽  
Bet Inhibitors ◽  
Acute Myeloid

Abstract Bromodomain and extra terminal proteins (BETs) act as non-oncogene addiction targets1,2. BET inhibitors (BETi) are epigenetic drugs being explored as a promising therapeutic avenue in cancer, especially acute myeloid leukemia (AML)3,4,5. Although early clinical trials have highlighted the efficacy of this first-in-class therapy, chemotherapy resistance, which is commonly emerged during drug treatment, still hurdle the prevalence of BETi6,7. To seek for a strategy to overcome this hurdle, we carried out a combinatorial drug screening and discovered a synthetic lethality via combined inhibition of BRD4 and CDK7. Cyclin-dependent kinase 7 (CDK7), acting as a member of the general transcription factor H (TFIIH), can directly target the carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNAPII for phosphorylation at serine 5 (Ser5) and serine 7 (Ser7), which is critical for transcription initiation. Recently, CDK7 has also been implicated in super-enhancer (SE) regulation8. Dual pharmacological inhibition of BRD4 and CDK7 caused a synergistic effect on the growth and apoptosis of K562 cells. Knockdown of BRD4 and CDK7 in the same cell line showed similar results as drug treatment, thus ruling out the off-target effects of inhibitors used in this study. Furthermore, we confirmed this synergistic effect in vivo using a xenograft mouse model. After 2 weeks of treatment, mice bearing BET-resistant cells showed prolonged lifespan with less leukemic burden and reduced leukemic blast infiltration in spleen, liver, and bone marrow when treated with combinatorial regimen compared with control and single agent groups. Furthermore, RNA-seq analysis showed that genes involved in cell cycle and proliferation were significantly down-regulated, while genes associated with apoptosis were up-regulated. Myc-associated genes were prominently altered, suggesting that c-Myc is one of the top targets of BRD4 and CDK7 inhibition. Ectopically expressing c-Myc partially rescued the synthetic lethality induced by BRD4 and CDK7 treatment, a finding further confirmed that Myc is potential targets of inhibition of BRD4 and CDK7. Further molecular analysis showed that BRD4 and CDK7 inhibition altered the RNAPII activity at actively transcribed genes. Interestingly, we also observed a strong reduction of RNAPII enrichment, but not H3K27Ac enrichment, at super-enhancers (SE). Loci-specific experiment on c-Myc SE regions will further reveal detailed mechanisms on how BRD4 and CDK7 inhibition affect RNAPII activity at SE to influence gene transcription. Congruently, by adopting a combinatorial drug screen approach, we identified the synthetic lethality of BRD4 and CDK7 inhibition in BRDi resistant leukemia cells both in vitro and in vivo. Our findings identify a new strategy that may enhance the clinical utility of BET inhibitors, especially for the treatment of BETi resistance. Refereneces 1. Dawson, M. A., Kouzarides, T. & Huntly, B. J. Targeting epigenetic readers in cancer. N. Engl. J. Med. 367, 647-657 (2012) 2. Shi, J. & Vakoc, C. R. The mechanisms behind the therapeutic activity of BET bromodomain inhibition. Mol. Cell 54, 728-736 (2014) 3. Herait, P. E. et al. BET-bromodomain inhibitor OTX015 shows clinically meaningful activity at nontoxic doses: interim results of an ongoing phase I trial in hematologic malignancies. Cancer Res.74, CT231 (2014) 4. Zuber, J. et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524-528 (2011) 5. Delmore, J. E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904-917 (2011) 6. Rathert, P. et al. Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature 525, 543-547 (2015) 7. Fong, C. Y. et al. BET inhibitor resistance emerges from leukaemia stem cells. Nature 525, 538-542 (2015) 8. E. Chipumuro, E. Marco, C.L. Christensen, et al. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer. Cell, 159, 1126-1139 (2014) Disclosures No relevant conflicts of interest to declare.

scholarly journals Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Chunliang Li ◽  
Shaela Wright ◽  
Jianzhong Hu ◽  
Yang Zhang ◽  
Judith Hyle ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Adaptor Protein ◽  
Leukemia Cells ◽  
Resistance Phenotype ◽  
Bet Inhibitors ◽  
Resistant Cells

Abstract MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood. We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression. In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. Disclosures No relevant conflicts of interest to declare.

Involvement of MCL1, c-myc, and cyclin D2 protein degradation in ponatinib-induced cytotoxicity against T315I(+) Ph+leukemia cells

2020 ◽  
Vol 525 (4) ◽  
pp. 1074-1080 ◽  
Author(s):  
Chisato Inoue ◽  
Sayaka Sobue ◽  
Yoshiyuki Kawamoto ◽  
Yuji Nishizawa ◽  
Masatoshi Ichihara ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Leukemia Cells ◽  
Cyclin D2 ◽  
D2 Protein

scholarly journals A Combination Strategy Targeting Enhancer Plasticity Exerts Synergistic Lethality Against Beti-Resistant Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5053-5053
Author(s):  
Lei Guo ◽  
Jia Li ◽  
Hongxiang Zeng ◽  
Anna Guzman ◽  
Tingting Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Rna Polymerase Ii ◽  
Conflicts Of Interest ◽  
Leukemia Cells ◽  
Cancer Drug ◽  
Distal Enhancer ◽  
Combination Strategy ◽  
Cancer Drug Resistance ◽  
Bet Inhibitors

Primary and acquired drug resistance is a major challenge to achieving optimized clinical outcomes during cancer treatment which can arise from transcription reactivation, bypass and alteration during anticancer treatment [1-3]. Epigenetic dysregulation is emerging as a crucial component involved in drug resistance. Transcriptional adaptation during drug treatment is often mediated by inducible histone modifications, especially histone H3 lysine 27 acetylation (H3K27ac) at the distal enhancer elements, thus activating the transcription of drug resistance-associated genes [4-6]. BRD4 (bromodomain-containing protein 4), a member of the bromodomain and extra-terminal domain (BET) family, acts as a chromatin reader to regulate transcription by linking histone acetylation and core components of the transcriptional apparatus [7]. BET inhibitors (BETi), as exemplified by JQ1 and I-BET151, have been shown to suppress the growth of multiple types of tumor both in vitro and in vivo [8]. However, drug resistance associated with BETi becomes one of the major hurdles hampering the clinical applications of these promising drug candidates [8, 9]. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on CRISPR interference, we identified the second intron of IncRNA, PVT1, as a unique bona fidegained enhancer that restored MYCtranscription independent of BRD4 recruitment. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYClocus in BETi-resistant leukemia cells. Furthermore, recipient mice transferred with BETi-resistant murine MLL-AF9 AML cells receiving the combination treatment showed the most effective therapeutic outcomes, as characterized by prolonged overall survival and reduced tumor burdens in the spleen and bone marrow. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies. References 1. Mansoori, B., et al., The Different Mechanisms of Cancer Drug Resistance: A Brief Review.Adv Pharm Bull, 2017. 7(3): p. 339-348. 2. Konieczkowski, D.J., C.M. Johannessen, and L.A. Garraway, A Convergence-Based Framework for Cancer Drug Resistance.Cancer Cell, 2018. 33(5): p. 801-815. 3. Holohan, C., et al., Cancer drug resistance: an evolving paradigm.Nat Rev Cancer, 2013. 13(10): p. 714-26. 4. Zanconato, F., et al., Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4.Nat Med, 2018. 24(10): p. 1599-1610. 5. Takeda, D.Y., et al., A Somatically Acquired Enhancer of the Androgen Receptor Is a Noncoding Driver in Advanced Prostate Cancer.Cell, 2018. 174(2): p. 422-432 e13. 6. Chen, X., et al., A novel enhancer regulates MGMT expression and promotes temozolomide resistance in glioblastoma.Nat Commun, 2018. 9(1): p. 2949. 7. Jang, M.K., et al., The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription.Mol Cell, 2005. 19(4): p. 523-34. 8. Andrieu, G., A.C. Belkina, and G.V. Denis, Clinical trials for BET inhibitors run ahead of the science.Drug Discov Today Technol, 2016. 19: p. 45-50. 9. Pervaiz, M., P. Mishra, and S. Gunther,Bromodomain Drug Discovery - the Past, the Present, and the Future.Chem Rec, 2018. 18(12): p. 1808-1817. Disclosures No relevant conflicts of interest to declare.

scholarly journals Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors

2017 ◽  
Vol 23 (9) ◽  
pp. 1046-1054 ◽  
Author(s):  
Hana Janouskova ◽  
Geniver El Tekle ◽  
Elisa Bellini ◽  
Namrata D Udeshi ◽  
Anna Rinaldi ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Cancer Type ◽  
Bet Inhibitors ◽  
Opposing Effects ◽  
Bet Protein

scholarly journals Development of an N-Terminal BRD4 Bromodomain-Targeted Degrader

2021 ◽  
Author(s):  
Anand Divakaran ◽  
Huda Zahid ◽  
Wenwei Lin ◽  
Taosheng Chen ◽  
Dan Harki ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Small Molecules ◽  
Ubiquitin Ligase ◽  
Ternary Complex ◽  
Selective Inhibitor ◽  
Selective Degradation ◽  
Ternary Complex Formation ◽  
Native Proteins ◽  
Bet Inhibitors ◽  
Ubiquitin Ligase E3

Targeted protein degradation is a powerful induced-proximity tool to control cellular concentrations of native proteins using small molecules. However, the design of selectivity in protein degradation remains challenging. In the case of Bromodomain and Extra-Terminal (BET) family proteins, BRD4 has emerged as the primary therapeutic target over other family members BRD2, 3 and T, but strategies to selectively degrade BRD4 rely on the use of pan-BET inhibitors optimized for BRD4:E3 protein-ubiquitin ligase (E3) ternary complex formation. Here, we report a potent and selective inhibitor for the first bromodomain of BRD4, iBRD4-BD1 (IC50 = 12 nM, 23-6200-fold intra-BET selectivity). We further use this novel inhibitor to develop dBRD4-BD1 that induces selective degradation of BRD4 at a DC50 of 280 nM. The design of BRD4 selectivity up-front enables the study of BRD4 biology in the absence of wider BET-inhibition, simplifies design of future BRD4-selective degraders as new E3 recruiting ligands are discovered, and provides a tool to design additional heterobifunctional BRD4-selective probes.

scholarly journals Bromodomain and Extra Terminal Domain (BET) Inhibitors Sensitize Chronic Myelomonocytic Leukemia (CMML) to PIM Inhibition Via Downregulation of Mir-33a

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4220-4220
Author(s):  
Christopher Letson ◽  
Alexis Vedder ◽  
Ariel Quintana ◽  
Phillip Liu ◽  
Brett Reid ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Bet Inhibitors ◽  
Dose Dependent

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

Ferritin-Conjugated Antibody for the Demonstration of Isoantigens on the Surface of Murine Cells

1968 ◽  
Vol 26 ◽  
pp. 48-49
Author(s):  
T. Aoki ◽  
J. Izard ◽  
U. Hämmerling ◽  
E. de Harven ◽  
L. J. Old
Keyword(s):  
Cell Surface ◽  
Gamma Globulin ◽  
Leukemia Cells ◽  
Labelling Technique ◽  
Direct Labelling ◽  
Labelling Method

Although a variety of viral and cellular antigens have been demonstrated by ferritin-labeled antibody, this technique has not been used to locate isoantigens on the surface of nucleated cells. The recognition of several systems of isoantigens on the surface of thymocytes, lymphocytes and leukemia cells of the mouse and the ease with which these cells can be obtained in free suspension led us to consider the ferritin-labelling method to determine the amount and location of these isoantigens on the cell surface. Because of the problems involved in the direct labelling of mouse gamma globulin by ferritin, we have chosen an indirect labelling technique (i.e. ferritin-conjugated rabbit anti mouse γG)to detect localization of mouse isoantibody.

The distribution of antigen on the surface of RBL-2H3 rat basophilic leukemia cells observed by back-scattered electron imaging

1986 ◽  
Vol 44 ◽  
pp. 274-275
Author(s):  
R.F. Stump ◽  
J.R. Pfeiffer ◽  
JC. Seagrave ◽  
D. Huskisson ◽  
J.M. Oliver
Keyword(s):  
Cell Surface ◽  
Dynamic Properties ◽  
Leukemia Cells ◽  
Antigen Binding ◽  
Scattered Electron ◽  
Ige Receptor ◽  
Receptor Complexes ◽  
Rat Basophilic Leukemia Cells ◽  
Electron Imaging ◽  
Basophilic Leukemia

In RBL-2H3 rat basophilic leukemia cells, antigen binding to cell surface IgE-receptor complexes stimulates the release of inflammatory mediators and initiates a series of membrane and cytoskeletal events including a transformation of the cell surface from a microvillous to a lamellar topography. It is likely that dynamic properties of the IgE receptor contribute to the activation of these responses. Fewtrell and Metzger have established that limited crosslinking of IgE-receptor complexes is essential to trigger secretion. In addition, Baird and colleagues have reported that antigen binding causes a rapid immobilization of IgE-receptor complexes, and we have demonstrated an apparent increase with time in the affinity of IgE-receptor complexes for antigen.

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