Editor's Note: In the high summer of 2025, the highly anticipated European Hematology Association (EHA) Annual Meeting was convened, bringing together top global experts in hematologic oncology to discuss cutting-edge progress and future directions in the field. In the Chronic Lymphocytic Leukemia (CLL) session, Dr. Daisy Diaz, a postdoctoral fellow from MD Anderson Cancer Center in the United States, representing the team of her mentor, Professor Deepa Sampath, delivered an exceptional presentation titled "A Study on Transcriptional Reprogramming and Survival Co-dependencies Following Venetoclax Resistance in Chronic Lymphocytic Leukemia." This research systematically unveiled the complex resistance mechanisms in CLL after the failure of Venetoclax treatment and proposed an innovative solution based on Proteolysis-Targeting Chimera (PROTAC) technology, offering a highly promising new approach for clinically overcoming dual-drug resistance.

Dual Resistance: A Grave Challenge in CLL Targeted Therapy

In recent years, targeted drugs represented by the BCL2 (B-cell lymphoma 2) inhibitor Venetoclax and BTK (Bruton’s tyrosine kinase) inhibitors have significantly improved the prognosis for CLL patients, ushering in a “chemo-free” era of treatment. However, as Dr. Diaz pointed out at the beginning of her presentation, the emergence of secondary resistance, particularly in “dual-refractory” patients who are unresponsive to both classes of drugs, has become a major challenge in current clinical practice. These patients have extremely limited subsequent treatment options and a very poor prognosis.

The mechanisms leading to resistance are intricate and exhibit high inter- and intra-patient heterogeneity. On one hand, the BCL2 or BTK genes themselves may undergo mutations that prevent drug binding; on the other hand, tumor cells can “bypass” the drug’s blockade by activating other signaling pathways or upregulating alternative survival proteins. Dr. Diaz mentioned that previous studies have confirmed that the overexpression of other anti-apoptotic proteins, such as MCL1, BFL1, and BCL-xL, is one of the key reasons for Venetoclax resistance. Faced with such a complex resistance network, the core objective of her team’s research became the search for a therapeutic target that could cover different resistance mechanisms and be universally applicable.

Multidimensional Analysis: Unveiling a New Pattern of Survival Co-dependency in Resistant CLL

To deeply investigate the intrinsic changes in dual-refractory CLL cells, the research team performed Whole Exome Sequencing and single-cell RNA sequencing on a sample cohort from 15 dual-refractory patients.

Genomic analysis revealed the complexity of the resistant population: although BCL2 mutations were detected in 6 patients, their tumor fraction was at most 16%, implying that the majority of resistant cells rely on non-mutational mechanisms. In contrast, mutations related to the BTK pathway (BTK or PLCG2) were more prevalent, persisting in up to 60% of the samples. Furthermore, about half of the patients carried high-risk TP53 mutations. This heterogeneity in the genetic background further underscores the necessity of finding a common therapeutic target.

The more critical findings came from single-cell transcriptomic analysis. By finely clustering the CLL cells, the research team identified several cell subpopulations that were significantly enriched upon relapse. Among them, two clusters (cluster 5 and 6) showed co-expression of BCL2 and another anti-apoptotic protein, MCL1, suggesting that cells may use this “survival co-dependency” to resist drugs that solely target BCL2. Another subpopulation (cluster 1), which was significantly expanded in several patients with TP53 mutations, was characterized by extremely low BCL2 expression but had newly acquired expression of BCL2A1 (which encodes the BFL1 protein). This finding suggests that mutant p53 may play an oncogenic role in driving resistance, although this still requires further functional validation.

Overall, pathway enrichment analysis pointed to a common “mastermind” — the NF-κB signaling pathway. This pathway was universally activated in relapsed, resistant cells and, as a key transcription factor, it can drive the upregulation of various anti-apoptotic genes (such as BCL2L1 encoding BCL-xL, MCL1, and BCL2A1). Proteomics data also confirmed that BCL-xL protein levels were significantly induced in all evaluated resistant samples. Together, these findings paint a clear picture: resistant CLL cells undergo transcriptional reprogramming to establish a “co-dependency” on multiple anti-apoptotic proteins, including BCL2 and BCL-xL, thereby building a robust survival defense.

Precise Targeting: A BCL2/BCL-xL Dual-Efficacy Degrader Paves a New Therapeutic Path

Based on the above findings, simultaneously blocking both BCL2 and BCL-xL would theoretically be an effective strategy to overcome resistance. However, traditional BCL-xL inhibitors (like Navitoclax) have limited clinical application due to their severe toxicity to platelets (platelets depend on BCL-xL for survival).

To solve this dilemma, the research team, in collaboration with partners, developed an innovative BCL2/BCL-xL dual-efficacy degrader, a novel molecule based on PROTAC technology. Dr. Diaz explained that this molecule connects Navitoclax to a VHL E3 ligase ligand via a linker. When it binds to BCL2 or BCL-xL, it recruits the VHL ligase to tag the target protein with “ubiquitination,” directing it for degradation by the proteasome system. This design cleverly leverages the differences in protein degradation efficiency among different cell types, aiming to achieve highly efficient clearance of the target proteins in tumor cells while avoiding toxicity to platelets. In vitro experimental data confirmed that the platelet safety of this degrader (codename WH in the report) was significantly improved.

In terms of efficacy validation, this novel degrader demonstrated potent anti-tumor activity. In CLL cells taken directly from clinically Venetoclax-resistant patients, the drug more effectively activated the apoptosis-executing proteins BAX and BAK and promoted the mitochondrial release of cytochrome c. Functional analysis showed that it could simultaneously degrade BCL-xL and inhibit or degrade BCL2, fundamentally dismantling the survival dependency of the resistant cells. Ultimately, its potency in inducing apoptosis (as measured by caspase-3 cleavage) was about 5 times higher than that of Venetoclax.

Even more excitingly, the degrader showed activity in resistant samples with different genetic backgrounds, including all tested BCL2 mutant, PLCG2 mutant, and some TP53 mutant samples. To further validate its efficacy against mutant proteins, the team used CRISPR technology to construct cell lines carrying different BCL2 resistance mutations. Experiments proved that the degrader could effectively degrade both wild-type and all mutant forms of the BCL2 protein in a dose-dependent manner.

Expert Dialogue and Clinical Translation Outlook During the Q&A session after the presentation, Professor Arnon Kater from Amsterdam asked whether future clinical trials should use assays like BH3 profiling to pre-select patients who are dependent on BCL2/BCL-xL. Dr. Diaz agreed, stating that as more drugs targeting different anti-apoptotic proteins enter development, transitioning functional assays from being validation studies in clinical trials to prospective screening tools will be a key step toward achieving precision medicine.

Meanwhile, in response to the moderator’s question about whether first-line combination therapy could prevent such resistance mechanisms, Dr. Diaz explained that her research cohort included patients who had progressed during combination therapy with BTK and BCL2 inhibitors. The cells from these patients were completely insensitive to Venetoclax in vitro. This indicates that for the “most difficult-to-treat” patient population that still progresses under potent combination therapy, there is an urgent need for therapies with entirely new mechanisms of action, such as the one presented in her talk.

Conclusion

Dr. Diaz’s presentation not only systematically depicted the complex network of transcriptional reprogramming and survival dependencies in dual-refractory CLL but, more importantly, demonstrated how an innovative drug based on PROTAC technology can “precisely treat the cause,” potentially solving the most challenging clinical problem in CLL therapy today. This research, spanning from mechanistic exploration to translational application, perfectly exemplifies the paradigm of basic research driving clinical breakthroughs. It also heralds that new technologies, represented by protein degradation, will play an increasingly important role in the future treatment of hematologic malignancies, bringing new hope to more patients with refractory and relapsed disease.