A-1331852

Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.

Our study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells. Graphical abstract created with BioRender.com, illustrating the workflow and summarizing main results.

Targeting Bcl-xL activity with a specific BH3 mimetic, A-1331852, blocked the induced neutrophil survival without impacting their normal lifespan...Finally, our human tumor data indicate the same role for Bcl-xL on pro-tumoral neutrophil survival. These results altogether provide preclinical evidence for safe neutrophil targeting based on their aberrant intra-tumor longevity.

MethodsA diverse panel of EBV+ T/NK lymphoma cell lines including SNK1, MECO4, SNK6, SNT8, SNK10, SNT15 and SNT16 were evaluated for sensitivity to MMAE alone and with specific BH3 family inhibitors including A1331852, inhibiting BCL-XL, venetoclax inhibiting and BCL-2 and the MCL-1 inhibitor S63845 (Generon). These data show the potential of BV with BCL-XL inhibition to be an effective and tolerable treatment for ENKTL. We are now planning a detailed preclinical study to better understand the efficacy and safety of this combination in vivo and progress towards a future clinical trial.

Notably, carboplatin and A-1331852 (Bcl-xL inhibitor) showed an additive effect in four of eight OC samples tested, while afatinib and A-1331852 led to synergy in five of seven OC models. In conclusion, our 3D DET3Ct platform can rapidly define potential, clinically relevant data on efficacy of existing drugs in OC for precision medicine purposes, as well as provide insights on emerging drugs and drug combinations that warrant testing in clinical trials.

In cells treated with JAK inhibitor ruxolitinib, we found that while BCL-xL expression levels decreased nearly 90% in ruxolitinib treated CALRdel52 cells, this decrease was much more modest in CALRins5 cells, suggesting JAK/STAT activation is only partially responsible for BCL-xL up-regulation in CALRins5 MPN cells. We observed that CALRins5 cells displayed decreased viability in response to A-1331852 alone and in combination treatments, suggesting an increased sensitivity to BCL-xL inhibition. In conclusion, we demonstrate that CALRins5-driven MPN cells display an enhanced sensitivity to BCL-xL inhibition, which may represent an effective therapeutic approach for CALRins5+ MPN patients.

Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity...Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

This shift was also reflected in an ex vivo drug treatment assay showing decreased sensitivity to the MCL-1 inhibitor S63845 and the BCL-XL inhibitor A-1331852 in ALL cells from VEN- treated mice compared to control- treated mice (S63845 EC50 1.5 vs. 2.2 µM, A-1331852 EC50 9.3 vs. 15.3 µM). Taken together, acquired VEN-resistance was recapitulated in a co-clinical trial model of BCP-ALL with repeated in vivo treatment cycles showing lower drug sensitivities along with increasingly reduced in vivo anti-ALL activity of VEN. Characterization of acquired VEN-resistance revealed decreased functional dependency on anti-apoptotic proteins and downregulation of pro-apoptotic BIM and BAX, thus pointing to an imbalance of pro- and anti-apoptotic molecules, which can be potentially targeted by directed compounds bypassing resistance to specific BCL-2 inhibition.

Furthermore, A-1331852 acted synergistically with the BCL2 inhibitor ABT-199 to induce U937 and ABT-199-resistant U937 cell death by inhibiting MCL1 expression. A similar phenomenon caused A-1331852-induced MCL1 downregulation and cytotoxicity in AML HL-60 cells. Collectively, our data suggest that A-1331852 shows an off-target effect of inhibiting MCL1 transcription, ultimately leading to U937 and HL-60 cell death.

Notably, Ifosfamide, auranofin, DMAPT, and A-1331852 emerged as potential therapeutic agents for GARS1-upregulated tumors. Our experimental findings strongly suggest that GARS1 promotes the proliferation and migration of bladder cancer cells. GARS1 holds promise as a potential prognostic marker and therapeutic target for pan-cancer immunotherapy, offering valuable insights for the development of more precise and personalized approaches to tumor treatment in the future.

We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

109 AML samples' blast-specific ex vivo responses to venetoclax (BCL-2 inhibitor), navitoclax (BCL-2/BCL-XL inhibitor), A-1331852 (BCL-XL inhibitor) and S-63845 (MCL-1 inhibitor) were profiled by flow cytometry... In the computational comparison analysis of VenEx trial participants' genetic aberrations and BH3 mimetic ex vivo responses, TP53 mutation (p=0.005) and complex karyotype (p=0.003) were the strongest predictors of venetoclax resistance, while IDH2 (p=0.006) and SRSF2 (p=0.007) mutations predicted favorable responses (Figure A)... This study showed that AML patients harboring TP53 mutations have reduced ex vivo sensitivity to venetoclax, which might be associated with decreased BCL-2 protein expression. In contrast, our BH3 mimetic drug screening results demonstrated that the dual inhibition of BCL-2 and BCL-XL by navitoclax is capable of inducing apoptosis in myeloid blast cells regardless of TP53 mutation status. However, larger patient cohorts and more extensive functional analysis of the anti/pro-apoptotic dependencies in this patient group is warranted.

Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumor; especially since the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

We used well-established HGSOC cell lines and human primary tumors to measure sensitivity to inhibitors of pro-survival BCL-2 family proteins as single agents and in combination with the front-line ovarian cancer chemotherapy drugs carboplatin and paclitaxel. Based on BH3 profiling, flow cytometry-based cell death analysis and colony formation assays, we found that HGSOC cells are particularly sensitive to BCL-XL inhibitors including the first-generation BH3 mimetics ABT-263 and A1331852 as well as novel proteolysis-targeting chimeras (PROTACs) that degrade BCL-XL...We also detected BCL-XL dependence in ovarian cancer tumorigenesis models utilizing human fallopian tube secretory epithelial cells, the putative cell of origin for HGSOC. Overall, we find that BCL-XL inhibitors, especially PROTACs, can safely enhance the chemosensitivity of HGSOC cells in vitro and in vivo and may therefore prevent tumor recurrence in women diagnosed with this disease.

In solid tumor lines other than SCLC, the combination of lurbinectedin with a specific BCL2L1 inhibitor (A-1331852) was also synergistic. Correspondingly in vivo, enhancement in the efficacy of lurbinectedin was observed in combination with venetoclax in SCLC models, while in solid tumor models other than SCLC the combination with a specific BCL2L1 inhibitor enhanced efficacy. Collectively, these data identify MCL1 as a specific lurbinectedin target and suggest that combinations that target other pro-survival proteins may represent a viable strategy to enhance the anti-tumor activity of lurbinectedin.

In vivo, the triple combination CM272, A1331852 (BCL-XL inhibitor) and an anti-PD-1 moAb significantly reduced tumor growth and increased overall survival in three subcutaneous lung cancer models (LLC, 393P, Lacun-3) in comparison to double treatments...The mechanisms underneath the antitumor responses include the modulation of the energy metabolism in tumor cells, leading to cell death boosted by an anti-BCL-XL pro-apoptotic drug, along with fostering the immune system to generate an efficient anti-tumor response assisted by ICB. This study reveals the potential of epigenetic therapeutics to treat and cure patients with solid tumors.

Methods Cell lines were treated with ADI-PEG20 and A-1331852 (Bcl-xL inhibitor) and monitored for cell death using the Incucyte. Bcl-xL inhibition synergies with ADI-PEG20 to induce apoptosis. This study provides the preclinical rationale for combining ADI-PEG20 with next generation Bcl-xL inhibitors such as the Bcl-xL PROTAC in a phase I clinical trial.

High-throughput screening of > 500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. Combining BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

We demonstrate that pan Bcl-2 family inhibition (ABT-263, rER: 1.52-1.56) or Bcl-xL specific inhibition (WEHI-539, A-1331852; rER: 1.31-2.00) radiosensitized wild-type PIK3CA/PTEN TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize mutant PIK3CA/PTEN TNBC (rER: 0.90 - 1.07; MDA-MB-468, CAL-51, SUM-159). In vivo, ABT-263 or A-1331852 in combination with RT decreased tumor growth and increased tumor tripling time (p < 0.0001) in PIK3CA/PTEN wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and RT in women with wild-type PIK3CA/PTEN wild-type TNBC at high risk for recurrence.

Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

Despite the available chemoimmunotherapy R-CHOP, one third of DLBCL patients still have primary refractory disease or relapse, and the incidence of these lymphomas continuously increases...Furthermore, co-treatment of Brusatol with inhibitors of Bcl-2 (Venetoclax), Bcl-XL (A-1331852), Mcl-1 (S63845), BTK (Ibrutinib), PI3K (Idealisib), Myc (EN4, JQ1), respectively, was performed in the same four cell lines and Annexin V levels were measured after 24h...Additionally, the combination of Brusatol with Venetoclax results in enhanced induction of apoptosis. Thus, our study suggests that Brusatol, alone or in combination with Venetoclax, represents a very interesting agent for further development of novel anti-lymphoma therapies.

The high-throughput screens identified BCL-XL-selective inhibitors including A-1331852 as highly effective against erythroid and megakaryoblastic leukemia cell lines in comparison with 15 cell lines representing other AML types (Figure B). Combining BCL-XL inhibition with the JAK inhibitor ruxolitinib, the BCL-2 inhibitor venetoclax, or the hypomethylating agent azacitidine showed potential for more durable responses both in a 3-day ex vivo assay as well as long-term treatments spanning over a month. Collectively, our results suggest targeting BCL-XL as a potential therapy option in erythroid and megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

In this study, the BCL-XL inhibitor A1331852, MCL1-inhibitor S63845, dual PI3K-mTOR inhibitor bimiralisib (PQR309), BMI-1 inhibitor unesbulin (PTC596), MEK-inhibitor trametinib (GSK1120212), and STAT3 inhibitor C-188-9 were assessed as single agents and in combination with venetoclax, for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. For the venetoclax and bimiralisib combination treatment, responders were enriched for IDH2 and FLT3 mutations, whereas non-responders were associated with PTPN11 mutations. The combination of PI3K/mTOR dual pathway inhibition with bimiralisib and BCL2 inhibition with venetoclax has emerged as a candidate treatment in IDH2- and FLT3-mutated AML.

All 7 CFZ resistant MM cell lines developed cross resistances to the MCL-1 inhibitors S63845 and AZD-5991...Moreover, a slight increase in sensitivity to venetoclax or A1331852 was observed in resistant OPM-2 or NCI-H929 and U266 cells, respectively...Exposure to ixazomib (IXA) upregulated BCL-xL and BAK in CFZ resistant cell lines... Our data show significant deregulation of BH3 proteins in CFZ resistant cell lines highlighting the importance of the apoptosis signaling pathway in PI resistance. Consistent deregulation of BCL-xL and BAK throughout CFZ and IXA exposed CFZ resistant cell lines suggests that this might not be a CFZ specific effect. BH3 profiling indicates a change in dependency on anti-apoptotic BH3 proteins in CFZ resistant vs.

Our studies uncovered an unexpected link between cell state and BCL-XL dependence in ccRCC. Therapeutic agents that specifically target BCL-XL are available. Our work justifies testing the utility of BCL-XL blockade to target, likely, a clinically aggressive subset of human kidney cancers.

For instance, the sensitivity to the BCL-2 inhibitor venetoclax is increased in PI resistant AMO-1 cells and a deregulation of BH3 proteins Noxa and MCL1 was noted in MM cell lines exposed to bortezomib...Results Viability assays indicate that all 7 tested carfilzomib resistant MM cell lines developed a cross resistance to the MCL-1 inhibitors S63845 and AZD-5991. Decreased sensitivity to the BCL-2 inhibitor venetoclax as well as to the BCL-xL inhibitor A1331852 was observed in resistant KMS12PE cells...This is further emphasized by the coherent deregulation of BCL-xL and BAK throughout carfilzomib exposed carfilzomib resistant cell lines when compared to their sensitive cell line variants. Moreover, BH3 profiling indicates a change in dependency on anti-apoptotic BH3 protein in carfilzomib resistant vs. sensitive MM cell lines.

Bcl-2 (ABT-199), Mcl-1 (S63845) and Bcl-x L (A1331852) inhibitors are simulated to predict cell viability and compared to experimental results. Using a combination of in silico and in vitro experiments, we show that a computational model can predict the sensitivity of a cell to targeted therapies. Future work will test the ability of the model to predict patient responses to targeted therapies in order to enable a personalised medicine approach.

AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia. Aims In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845)...Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo . Conclusion In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL.

Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.

U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor...In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

By selectively inhibiting BCL-2, BCL-x, or MCL-1 (i.e. ABT-199, A-1331852, S63845) they shift the balance of pro- and anti-apoptotic proteins in favor of apoptosis. Interestingly, NOXA played a different role in both treatments, as genetic silencing of NOXA significantly rescued from JQ1/A-1331852-mediated apoptosis but not from JQ1/S63845-mediated apoptosis. In summary, JQ1/A-1331852 and JQ1/S63845 co-treatment represent new promising therapeutic strategies to synergistically trigger mitochondrial apoptosis in RMS.

Furthermore, ulixertinib treatment slowed tumor growth and significantly increased survival in NSG mice with orthotopic BT40 xenografts.Ulixertinib showed indications for anti-proliferative synergy in vitro, according to the Loewe and Bliss independence models, in combination with MEK inhibitors (trametinib, binimetinib) or senolytics (navitoclax, A1331852). Combinations with chemotherapy (carboplatin, vinblastine) were at most additive...The combination of ulixertinib with navitoclax was further investigated in the BT40 PDX mouse model, where tumor growth and survival were comparable to ulixertinib monotherapy.In conclusion, our data indicate a strong potential for ulixertinib as a clinically relevant therapeutic option for the treatment of pLGG to be further investigated in upcoming clinical trials. Potential synergism with MEK inhibitors and senolytics was noted and warrants further investigation.

Some of the most common responses were seen for the BCL-XL inhibitor A-1331852 (9/15 patients), Topotecan (7), Dactinomycin (7), Omipalisib (6) and Omacetaxine (6). Combination screening revealed that Carboplatin and A-1331852, a BCL-XL inhibitor, showed increased efficacy in 3 of the 5 tested patient samples. In conclusion, our 3D HT-drug testing assay DETECT with a combination screening capability could in the future be useful for guiding individualized treatment in a clinical setting as well as for identifying existing and emerging drugs and drug combinations for repurposing in OC.

IC50s were determined for afore mentioned cell lines using MTS assay for Temozolomide (TMZ, alkylating agent), Bcl-2 inhibitor ABT199, Bcl-xL inhibitor A1331852, Mcl-1 inhibitor AZD5991, and BTK inhibitor Ibrutinib. Our preclinical data suggests synergism of Bcl-2 protein inhibitors in GBM cells, both TMZ-naïve and TMZ-resistant. Our ongoing research is investigating the biological relevance of Bcl-2 in conjugation with other tumor suppressor proteins, in vitro and in vivo.

Co-inhibition of MCL-1 and BCL-2 with MCL-1 selective inhibitor S63845 and BCL-2 selective inhibitor ABT-199 inhibited NPC cell proliferation but the effect on cell viability was more profound with co-inhibition of MCL-1 and BCL-XL with S63845 and A-1331852, implying that MCL-1 and BCL-XL are crucial for NPC cell survival. Deletion of BFL-1 sensitized NPC cells to A-1331852 suggesting that BFL-1 may play a role in NPC cell survival. Taken together co-inhibition of BCL-XL and MCL-1/BFL-1 could be potential treatment strategies for NPC.

Our results indicated that NA-2a was selectively enriched in mitochondria transported by organic-anion-transporting polypeptide (OATP) transporters, which altered the permeability of the mitochondrial membrane, thereby promoting the entrance of A-1331852 to mitochondria and enhancing its disruption of the BIM-BCL-X complex, which finally led to the increased anticancer activity in vitro and in vivo. Collectively, our data provided overwhelming evidence that the combination of NA-2a and A-1331852 could be used as a promising synergistic therapeutic agent in NSCLC therapy.

Treatment with either the Bcl-X inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-X or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

The compounds S63845, ABT-199 (Venetoclax) and A-1331852 were used to inhibit MCL-1, BCL-2 and BCL-X L pro-survival proteins respectively. BCL-X L and MCL-1 are upregulated in MM cells by stromal interaction, and also in response to CFZ. The upregulation of pro-survival proteins by stroma may mediate MM cell resistance to PIs, and blockade of both BCL-X L and MCL-1 is required in order to abrogate the protective effect of stroma and restore sensitivity to CFZ. Combination of BH-3 mimetic drugs at subtherapeutic doses with proteasome inhibitors may be a potential therapeutic strategy.

The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo . Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation.