Overcoming Stress Response-Induced Drug Resistance through Targeting CDK9 in Ph+ Leukemia
Authors: Vogt Melina , Kemkes Marie , Bilen Berna-Selin , Schliehe-Diecks Julian , Scharov Katerina , Tu Jia-Wey , Fischer Ute , Pandyra Aleksandra A. , Borkhardt Arndt , Bhatia Sanil 
Published: December 2024

Introduction:

The toxicity and quality of life burden following polychemotherapy protocols are significant, even among pediatric ALL patients classified as “low risk.” Therefore, it is essential to look for alternative therapies for patients with high-risk features, such as Philadelphia chromosome-positive (Ph+) leukemia, where harsh treatments are usually required. Heat shock protein 90 (HSP90), involved in stress response, is a critical target in leukemia due to its significant upregulation and essential role in stabilizing numerous oncogenic proteins, such as BCR::ABL1 kinase. However clinical approval of HSP90 inhibitors (HSP90i) is hampered due to resistance mechanisms, such as the heat shock response (HSR). Our recent findings identified HSP90α as the principal contributor to resistance in Ph+ leukemia cells. The combination of CDK7i and HSP90i synergistically inhibited the HSR via RNA Polymerase II (RNAPII) in Ph+ leukemia cells resistant to multiple TKIs.1

Methods:

The present study investigated the effects of HSP90 and CDK9 inhibitors on Ph+ leukemia cells. Conventional and capillary-based (JESS) Western blotting assessed HSR- and apoptosis-related protein expression following HSP90i and CDK9i treatment. Isogenic cell line models overexpressing anti-apoptotic proteins MCL-1, BCL-2, and BCL-xL were generated. Time-dependent qPCR validated transcriptional changes in HSR- and apoptosis-related genes post-treatment. Matrix synergy drug screening was performed to identify synergistic drug interactions in Ph+ leukemia cell lines and patient-derived xenograft (PDX) leukemia cells.

Results:

We investigated the pivotal role of CDK9, a crucial mediator in the transcriptional elongation of stress response genes and downstream of CDK7 in regulating RNAPII mediated transcription. Concurrent inhibition of CDK9 and HSP90 effectively diminishes HSP90AA1 expression, a key chaperone in cellular stress responses. The co-targeting strategy disrupted the induction of the HSR by specifically downregulating HSP90α (p = 0.0003) and HSP70 (p &lt; 0.0001) at the protein level, along with downregulating HSP90α/β, HSP70, HSP40, and HSP27 at the transcriptional level. A notable consequence of this dual inhibition is the downregulation of short-lived oncogenic proteins such as c-Myc and Mcl-1 over a time period of 1-24 hours. Additionally, other anti-apoptotic members of the BCL-2 family, including BCL-2 and BCL-xL are also downregulated within 6-24 hours after the treatment. This reduction in anti-apoptotic proteins correlates with an increase in cleaved Poly (ADP-ribose) polymerase (cl-PARP) and apoptotic cells (% sub-G1; pHSP90i:Combi &lt; 0.0001; pCDK9i:Combi = 0.0001). Notably the combination of CDK9 and HSP90 inhibition is equally effective in isogenic leukemia cell line models, overexpressing BCL2 family members (BCL2, BCL-xl, and MCL-1). The synergistic efficacy of CDK9i and HSP90i has been validated across various leukemia cell lines (maximum ZIP score: &gt;15; N=12) and patient-derived leukemia cells (N=3), highlighting its potential therapeutic significance in combating resistance.

Summary:

Dual targeting of HSP90 and CDK9 achieves HSR abrogation through RNAPII inhibition, leading to disruption of the transcription machinery responsible for activating stress response genes. Further, the combination will be tested in preclinical leukemia models, both alone and in conjunction with standard chemotherapy, with the goal of reducing doses and achieving stable remissions.

1. Vogt, M.; Dienstbier, N. et al. Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells. Cell Death & Disease 2023, 14 (12), 799.