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.

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.

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.

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.

Preferable safety profiles of FCN-683 were shown with no potential hERG inhibitory effect and less drug-drug interaction potential, as evidenced by no inhibitory effect (IC50 >50 μM) on CYP2C9 enzyme compared with VEN (IC50 1.05 μM). Together, FCN-683 is highly potent, selective and highly efficacious against a variety of clinically relevant VEN-resistance BCL-2 mutations in vitro and in vivo and exhibits favorable PK and safety profiles, highlighting its therapeutic potential to become an effective therapeutic approach for VEN-naïve or -resistant BCL-2-addicted B-cell malignancies.

L105 showed great potency on wild type and acquisition resistance mutations of Bcl-2, excellent oral bioavailability, and superior preclinical anti-tumor activities and safety profiles. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers.

Further interrogation of differential gene expression following SpiD3 treatment in VEN-resistant cells is ongoing to delineate the molecular mechanism underlying SpiD3's anti-tumor properties in VEN-resistant CLL. In summary, our data (a) supports the efficacious combination of SpiD3 with VEN as a viable therapeutic approach in CLL to bypass NF-κB activation underlying VEN resistance and (b) introduces SpiD3 as an alternative therapeutic approach for VEN-resistant CLL patients.

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

In summary, HZ-L105 as a next generation Bcl-2 inhibitor, showed great potency on wild type and acquisition resistance mutations of Bcl-2 in vitro, excellent oral bioavailability, superior anti-tumor activities on preclinical xenograft model, and safety profiles,. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers.

Treatment for chronic lymphocytic leukemia (CLL) is primarily based upon the use of small molecules targeting Bruton's tyrosine kinase (BTK; ibrutinib, acalabrutinib) or BCL-2 (venetoclax)...The efficacy of WH2544 and PZ18753b in treating naïve CLL cells fell in between that of venetoclax and navitoclax...In conclusion, our results suggest that venetoclax refractory CLL cells retain survival dependency on BCL-2 and BCL-xL proteins to evade apoptosis. This vulnerability can be therapeutically exploited using the BCL-2/BCL-xL dual targeting PROTACs PZ18753b and WH2544 in CLL.

Bcl-2 is a well-validated target for B cell malignancies as demonstrated by a selective Bcl-2 inhibitor venetoclax approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML). Our efforts may provide the opportunity to develop a novel Bcl-2 inhibitor which presents more benefits in CLL patients as well as other Bcl-2 mediated malignancies. BGB-11417 is currently undergoing phase1/2 clinical assessment in monotherapy or combination.

Methods Peripheral blood samples from 71 patients treated with venetoclax-rituximab or first line venetoclax-obinutuzumab combination were collected from 11 Hungarian oncohematological centres. In patients harboring multiple BCL2 mutations, convergent evolution of the CLL subclones may contribute to the driver mechanisms of resistance, justifying the comprehensive approach for the detection of these variants. In secondary venetoclax resistant cases displaying wild type BCL2 , further molecular screening methods are required to reveal alternative genetic or non-genetic reasons for disease progression.

In conclusion, this study shows that approximately one-third of pts on continuous venetoclax for 2+ years develop evidence of low-level BCL-2 G101V mut. Further work is underway to identify additional co-existing muts in BCL2 or other genes, and to characterize the additional genetic events at the time of clear clinical progression.

The BCL-2 inhibitor venetoclax has shown impressive efficacy in patients with chronic lymphocytic leukemia, but its clinical benefits are limited by acquired BCL-2 gene mutations that confer drug resistance. Further, lisaftoclax plus alrizomadlin more effectively blocked cell cycle entry into the G2/M phase, resulting in accumulation of sub-G1 apoptotic cells. In summary, our study demonstrates, for the first time, that co-targeting BCL-2 and MDM2-p53 apoptosis pathways overcame resistance to BCL-2 inhibitors conferred by acquired BCL-2 gene mutations, potentially offering a viable strategy to overcome drug resistance and thus providing a rationale for clinical investigation.

Our results show that exposing primary CLL samples (n=4) from treatment-naïve patients to the Bcl-xL degrader DT2216 is associated with apoptosis at concentrations known to be non-toxic to platelets (EC50 = 162 nM at 18 hr treatment). However, venetoclax-resistant CLL cells undergo a shift in dependence to alternative Bcl-2 family proteins, such as Bcl-xL and Mcl-1, as a mechanism for resistance to apoptosis. Thus, resistant CLL represents an excellent setting in which to continue testing the efficacy of these potent Bcl-xL degraders, to overcome resistance to Bcl-2 inhibitors.

Some benefits of PROTACs include the ability to engage with wild type and mutated proteins, as seen with BTK PROTACs degrading mutated ibrutinib, and the fact that PROTACs function through a catalytic mechanism implies less overall drug would need to be administered for the required therapeutic effect, this in turn may enhance the therapeutic window and permit a range of combination therapies that might not otherwise have been tolerated. Specific to venetoclax, we plan on attaching various linkers by modifying the solvent-exposed tetrahydropyran which will then be capped with E3 ligase ligands including cereblon-specific thalidomide ligand. These PROTACs will then be tested against wild type Bcl-2, G101V mutant Bcl-2, and venetoclax-sensitive and venetoclax-resistant AML cell lines.

Rare phenotypic variants of RS do occur under the treatment of ibrutinib or venetoclax, and genetic factors leading to RS are similar to those identified in patients treated with chemoimmunotherapy. To our best knowledge, we have reported the first BCL2 G101V mutation in an RS patient treated with venetoclax.

Potential benefits of PROTACs relative to the parent drug include: (1) they may be more refractory to point mutations, as observed 189 with a PROTAC-driven rescue of BTK mutational resistance to ibrutinib, and (2) the PROTAC mechanism is catalytic meaning lower doses for a therapeutic effect which could widen the scope for safe combination therapies as well as directly counter upregulationmediated resistance. We aim to modify the solvent-exposed tetrahydropyran with various linkers tethering E3 ligase ligands, including the cereblon-specific thalidomide ligand. All PROTACs will be tested against wild-type and G101V mutant BCL-2 proteins, as well as in venetoclax-sensitive and resistant AML cells.

RNA-seq analysis focusing on the patients with del 8p shows significant downregulation of the TNFRSF10A/10B genes (TRAIL-Rs), with gene set enrichment analysis (GSEA) showing positive enrichment for WNT5A/FZD4 signaling and CREB signaling via the PKC and MAPK pathways, concomitant with downregulation of the BCR and FCGR pathways at progression. Our data suggest several mechanisms of venetoclax resistance in CLL, including loss of TNFRSF10A/B, sometimes with MCL1 upregulation, as well as WNT pathway upregulation and BCR pathway downregulation, which we are now validating in primary patient PBMCs and relevant cell line models.

Strong and persistent tumor regression in xenograft models of lymphoid malignancies in mouse and rat were observed at well tolerated doses following weekly IV administration of S65487 in combination with the MCL-1-specific inhibitor, S64315/MIK665. Altogether, these data demonstrate that S65487/VOB560 has significant therapeutic potential against human lymphoid and myeloid malignancies as well as in patients with Venetoclax resistant leukemias. Clinical studies are currently ongoing with S65487/VOB560 (NCT03755154).

Ex vivo drug treatments included: BCL2i (inhibitor): venetoclax; MCL-1i: AZD5991, S63845 and BCLXLi: A133...No mutations in EZH2, CREBBP or EP300 were found... 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. Our data suggest that Bcl-2 inhibition should be favored over Mcl-1 inhibition in BCL2-R CLL.