BCL2 G101V

The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.

In summary, sonrotoclax emerges as a potential second-generation BCL2 inhibitor for the treatment of hematologic malignancies with the potential to overcome BCL2 mutation-induced venetoclax resistance. Sonrotoclax is currently under investigation in multiple clinical trials.

Venetoclax is a specific inhibitor of Bcl-2, the key protein which protects CLL cells from intrinsic apoptosis, whereas the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib kills CLL cells via blockade of B-cell receptor (BCR) signalling. In conclusion, WH25244 is a PROTAC-based Bcl-2/Bcl-xL degrader with the potential to overcome venetoclax-resistant CLL dependent on Bcl-xL and mutant Bcl-2. Relative to its precursor, navitoclax, it shows increased potency against CLL cells and decreased toxicity against platelets in vitro, due to its VHL-dependent activity and minimal expression of VHL in platelets.

Together our results demonstrate that iPSC-NK cells can be engineered to generate venetoclax-resistance for use in combination with concurrent venetoclax therapy to markedly improve treatment of AML. Furthermore, this work demonstrates that novel drug resistance mechanisms can be introduced via genome engineering into iPSC-derived NK cells as a new strategy to produce improved cell products for "off-the-shelf" therapy.

To evaluate if combination treatment of AZD0466 with BTK inhibitors would improve efficacy, we transplanted murine Eµ-TCL1 tumors into syngeneic recipient mice and randomized them for treatment with vehicle, ibrutinib (30mg/kg in drinking water), acalabrutinib (25mg/kg, p.o. QD), AZD0466 (70mg/kg, i.v., QW) and combination of AZD0466 with ibrutinib or acalabrutinib. Moreover, AZD4320 was highly efficacious in MAVER-1 and MINO cell line models where resistance to venetoclax mediated by BCL-XL upregulation was modelled by an in vitro dose escalation method. In summary, our pre-clinical study shows that the dual BCL2/BCL-XL inhibitor could represent an important treatment option for venetoclax resistance mediated by specific BCL2 mutations or BCL-XL upregulation and that its efficacy could be further improved upon combination treatment with BTKi.

Knowing that BCL-xL inhibition in the clinic has been limited by platelet toxicity, PROTACs PZ18753b and WH25244 were synthesized from navitoclax (BCL-2/BCL-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets. PZ18753b and WH25244 have preclinical efficacy in baseline CLL and can degrade, both, Bcl-xL and mutant Bcl-2 proteins, which are known to confer venetoclax resistance. This translational study supports the development of novel therapeutics for the treatment of CLL subgroups with adverse clinical prognosis and will open new frontiers as we better understand the biology of this disease.

We retrospectively analyzed gene mutations among venetoclax treated pts and showed that ASXL1 and NOTCH1 were frequently mutated in the resistant cohort. We also evaluated BCL2 G101V mutations using ddPCR and demonstrated that SF3B1 gene mutations were highly co-mutated with BCL-2. Notably, copy number loss of 8p is related to venetoclax resistance and the majority of these events are caused by unbalanced translocations in the context of highly complex karyotype.

Ex vivo drug treatments included: BCL2i (inhibitor): venetoclax; MCL-1i: AZD5991, S63845 and BCLXLi: A133. The genomic landscape of BCL2-R CLL is characterized by a high frequency of trisomy 12, subclonal NOTCH and RAS pathway mutations, as well as BCL2 and MLL2 mutations. Protein expression, BH3 profiling and viability assays data are consistent with nearly exclusive dependence on Bcl-2.

Patients previously treated with B-cell receptor pathway inhibitors (BCRi), such as ibrutinib and idelalisib, have lower response rates to Ven; those who are refractory to prior BCRi have a significantly higher risk of relapse than those who are not refractory [1]. With extended follow-up, Ven demonstrated durable responses in patients with CLL who progressed on BCRi, irrespective of BTK mutation status. BCL-2 resistance mutations were detected in 19.1% (13/68) of patients, generally at low VAF. These acquired mutations were detectable in patients with prolonged Ven exposure, supporting further exploration of strategies focused on time-limited Ven exposure.

After the first induction cycle with the 7+3 scheme (cytarabine + idarubicin), refractoriness was observed, in addition to the appearance of 6% of nuclei with BCL2 amplification by FISH, while maintaining the TP53 variant (confirmed by PCR). A new therapeutic scheme was initiated with decitabine 10- venetoclax... Clonal evolution of the leukemia was evidenced by the acquisition of BCL2 amplification alongside changes in the karyotype after antineoplastic treatment, and particularly following venetoclax administration, while maintaining the primary TP53 pathogenic variant.The increasing use of targeted therapies is improving remission and survival rates in most hematologic neoplasms, but it is also leading to the emergence of therapy-related clonal selections, as seen in this case, which could cause resistant relapses or even refractoriness. Understanding the mechanisms responsible for these phenomena would help to understand their relevance in the evolution of these patients.

To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

Of the tested antiapoptotic proteins, Bcl-xL overexpressing CAR T cells proved superior, having higher proliferation and increased anti-tumor activity in combination with or without BH3 mimetics, providing a new strategy to optimize CAR T cell function for the treatment of leukemia and lymphoma.