BRD4 is a common therapeutic target that can disrupt the Notch1, Myc, and CD44 pathways to effectively eliminate the T-ALL LICs

The NOTCH1-MYC-CD44 axis integrates cell-intrinsic and extrinsic signaling to ensure the persistence of leukemia-initiating cells (LICs) in T-cell acute lymphoblastic leukemia (T-ALL) and we provides evidence that BRD4 is a common therapeutic target to effectively eliminate the T-ALL LICs.
Published in Cancer
BRD4 is a common therapeutic target that can disrupt the Notch1, Myc, and CD44 pathways to effectively eliminate the T-ALL LICs
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Despite high rates of remission with current frontline therapy, a large proportion of patients with T-ALL, particularly adults, experience relapses with dismal outcomes, with less than 10% of patients surviving over the long term 1,2,3. LICs represent a reservoir of T-ALL and are believed to drive relapse and treatment resistance of this disease4. Notch1, Myc, and CD44 are implicated to have a role in the persistence of these LICs in T-ALL cases5,6,7. Although phenotypic definition of LICs in the T-ALL patients is imprecise, CD34+CD7+ cells are enriched in LIC compartment8. T-ALL LICs are functionally better characterized by high CD44 expression and low reactive oxygen species (ROS) levels6,9,10  . Reactive oxygen species (ROS) mitigation is essential for survival of T-ALL LICs11 , and a strategy to impair ROS mitigation through modulation of expression of CD44 and its variants, could help eliminate LICs. CD44 upregulation also contributes to leukemogenesis in T-ALL patients6 and has been identified as a NOTCH1 transcription target. Substantial commonalities exist in pathways activated downstream of NOTCH1 and MYC in T-ALL2. Mutated Notch1 co-occupies the distal enhancer region of the MYC promoting activation of NFkB signaling, Hes1, PTEN and PI3K/Akt pathways in a feed-forward loop circuit that supports leukemia cell growth, proliferation, and self-renewal7,12,13  .Thus, MYC inhibition could represent a powerful therapeutic strategy to treat T-ALL with Notch1 mutation/activation. It has been difficult to target MYC directly, but MYC can be epigenetically downregulated by the disruption of members of the bromodomain and extra terminal domain (BET) protein family, enriched in large enhancer complexes (termed “super-enhancers”) 14,15,16. BRD4, a BET family protein, binds to acetylated lysine residues in histone H3 and provides the scaffold to assemble multi-molecular super-enhancer complexes that drive expression of oncogenes, including Myc and anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-115,17,18 . ARV-825 is a proteolysis-targeting chimera (PROTAC) with three components: a thienodiazepine-based BRD4 ligand, a linker, and a cereblon-binding ligand. This chimera captures a BRD4 molecule and causes its proteasomal degradation via the E3 ligase cereblon. In corollary to this, we reported that sustained degradation of BRD4 led to downregulation of CD44, MYC, and CXCR4 in acute myeloid leukemia (AML) stem cells and improved survival in a mouse model of AML19.

Recent therapeutic efforts for T-ALL have relied heavily on targeting mutant Notch1 and its activating events with gamma secretase inhibitors and have been limited by on-target toxic effects20. In contrast, our work with the BRD4 degrader ARV-825 is focused on down regulation of NOTCH1 targets and cell-intrinsic pro-survival and/or antiapoptotic proteins as well as interaction with BM microenvironment interactions. To obtain a global view of the transcriptional changes in T-ALL with the BRD4 degrader, here we performed genome-wide gene expression profiling (GEP) of T-ALL cells .ARV-825 treatment resulted in down regulation of 729 genes and up regulation of 456 genes at a significant level (p ≤ 0.01) for a one-fold change in the log2 value for treatment group. Gene set enrichment analysis (GSEA) using gene signatures from the Molecular Signatures Database highlighted the down regulation of Myc target genes along with gene sets representing other oncogenic pathways: cell-cycle progression, hypoxia response, metabolism, and Notch pathway activity. Mechanistically, the data from the present study link degradation of BRD4 with transcriptional down regulation of CD44 and its variants, increasing oxidative stress. Using conditional Pten-deficient T-ALL mouse model and NOTCH1-mutated disseminated T-ALL PDX models, which recapitulate several features of human T-ALL biology, we showed that ARV-825 has single agent anti-leukemic activity and improves the survival in mice with T-ALL. Indeed, single-cell proteomic analysis of BM cells of ARV-825–treated mice using CyTOF revealed marked down regulation of NOTCH1, MYC, and CD44 along with a significant quantitative decrease in the phenotypically defined LIC population. Furthermore, we demonstrated that reduction in the overall LIC population resulted in extended survival of mice after secondary transplantation . Although the genetic mouse model used in our study has a Pten deletion, these mice often have secondarily acquired activating NOTCH1 mutations21. Our findings show the common role of BRD4 in the NOTCH1-, MYC-, and CD44-regulatory axis and we propose that BRD4 is a single target that can be used to disrupt all these pathways simultaneously eliminating T-ALL LICs.

Although we identified BRD4 degradation as a therapeutic means of eliminating T-ALL LICs, our gene array data raise concerns about upregulated CXCR4, other microenvironment niche molecules, and Wnt/b-catenin signaling after BRD4 degradation. These findings may point to potential mechanisms of resistance for bromodomain and extraterminal domain degraders when tested for treatment of T-ALL in clinical trials. Our preliminary work provides guidance towards the use of therapeutic combinations, including inhibition of Wnt/b-catenin signaling or CXCR4 expression to overcome these potential resistance mechanisms.

In summary, the present study provides evidence that BRD4 is a common therapeutic target that can disrupt the Notch1, Myc, and CD44 pathways to effectively eliminate the T-ALL LICs.

References

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Cancer Biology
Life Sciences > Biological Sciences > Cancer Biology
  • Leukemia Leukemia

    This journal publishes high quality, peer reviewed research that covers all aspects of the research and treatment of leukemia and allied diseases. Topics of interest include oncogenes, growth factors, stem cells, leukemia genomics, cell cycle, signal transduction and molecular targets for therapy.