When analyzing potential therapeutic strategies, we observed a stronger senolytic activity in these melanoma cell lines when specifically targeting BCL-xL using A-1331852, navitoclax or the PROTAC BCL-xL degrader DT2216. Furthermore, we identified that the main apoptotic inhibition was shaped by BCL-xL and BAK binding increase that prevented mitochondrial permeabilization and apoptosis. To our knowledge, this is the first time that the molecular basis for BCL-xL anti-apoptotic adaptation in senescence is described, paving the way for the development of new molecules that either prevent HRK downregulation or displace BCL-xL binding to BAK to be used as senolytics.

The cytotoxicity of trametinib (MEK inhibitor), DT2216 (BCL-xL degrader) and afatinib (pan-EGFR inhibitor) and their combination was tested in patient-derived, primary PDAC cells using a live cell imaging system. Pathway analysis revealed the addition of afatinib in triplet regimen further inhibited PI3K/AKT effectors of p90RSK, p70S6K, and GSK3α/β along with a secondary pathway of P38 MAPK. Our study identifies an important contribution of EGFR inhibition to elevate the response of PDAC, supporting a clinical assessment of this triplet combination in patients.

P1/2, N=81, Not yet recruiting, Children's Oncology Group

In summary, the combination therapy of SIAIS361034 and sorafenib represents an effective and safe approach for inhibiting hepatocellular carcinoma progression. The novel balancing mechanism may also provide insights for combination and precision therapies in the treatment of hepatocellular carcinoma.

In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. At this dosage, 753b was well tolerated in mice, without observable induction of severe thrombocytopenia as seen with navitoclax, and no evidence of significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients, warranting clinical trials in future.

P1, N=20, Completed, Dialectic Therapeutics, Inc | Recruiting --> Completed | Trial completion date: Mar 2024 --> Nov 2023

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.

However, the clinical utility of navitoclax, a BCL-xL and BCL-2 dual inhibitor, as demonstrated in combination with JAK2 inhibitor ruxolitinib in patients with myelofibrosis (Harrison et al...In this study, we evaluated the pre-clinical efficacy of DT2216 in combination with ruxolitinib, 5-azacytidine (AZA), or MCL-1 inhibitor S63845 in JAK2-mut AML models...CONCLUSIONS These findings highlight the promising efficacy of DT2216 in JAK2-mut AML, as evidenced by reduced cell viability, on-target degradation of BCL-xL, and synergistic anti-leukemia effects when combined with AZA, ruxolitinib or MCL-1 inhibitor. These results provide valuable insights into future therapeutic strategies for the treatment of JAK2-mut AML, particularly in the context of post-MPN AML.

BCL-2-targeting small molecule inhibitor venetoclax won FDA approval for chronic lymphocytic leukemia and acute myeloid leukemia...We reasoned that since earlier BCL-XL degraders used strong BCL-XL inhibitor as warhead, e.g. the warhead for DT2216 is ABT263, they could readily bind to and inhibit BCL-XL in PLT causing thrombocytopenia...In vitro toxicity profiling of NXD02 including Ames test and safety panel revealed no concerns so far. In vivo safety assessment is ongoing for NXD02 as a promising candidate for clinical development in liquid and potentially select solid tumors.

While CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with Venetoclax, they were vulnerable to strategies that target the megakaryocytic pro-survival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor Navitoclax and DT2216, a selective BCL-XL PROTAC (proteolysis-targeting chimera) degrader developed to limit thrombocytopenia in patients. Navitoclax or DT2216 treatment in combination with low dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.

P1, N=24, Recruiting, Dialectic Therapeutics, Inc | Trial completion date: Jun 2023 --> Mar 2024 | Trial primary completion date: Apr 2023 --> Dec 2023

We analyzed 13 sPCL for their sensitivity to BH3 mimetics targeting either BCL2 (venetoclax) or BCLXL (A1155463) and showed that 3 sPCL were efficiently killed by venetoclax and 3 sPCL by A1155463. In conclusion, our study demonstrated that patients with sPCL addicted to BCLXL, a small but a very challenging group, could potentially receive therapeutic benefit from DT2216. Clinical trials of DT2216 in this subset of sPCL patients are warranted.

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.

The inhibitors targeting G12D mutation (such as MRTX1133) have been recently developed, whereas those targeting other mutations are still lacking...In addition, we recently demonstrated that the combination of sotorasib with DT2216 (a BCL-X-selective degrader) synergistically inhibits G12C-mutated pancreatic cancer cell growth in vitro and in vivo...This chapter will review KRAS biochemistry, signaling pathways, different mutations, emerging KRAS-targeted therapies, and combination strategies. Finally, we discuss challenges associated with KRAS targeting and future directions, emphasizing pancreatic cancer.

P1, N=24, Recruiting, Dialectic Therapeutics, Inc | Trial primary completion date: Oct 2022 --> Apr 2023

In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-X and MCL-1.

Resistance to DT2216 is rare in a wide variety of T-ALL cells but when it occurs is correlated with decreased BCL-XL degradation. Resistance to DT2216 in T-ALL is not predicted by initial BCL-XL or BIM protein levels, or BCL-2 or MCL-1 levels before or after treatment. These data imply that a phase 2 clinical trial of DT2216 in T-ALL should be widely available and not limited to a subset of patients.

We first evaluated the sensitivity of 24 genetically diverse leukemia cell lines (17 AML, 5 T-ALL, and 2 AML secondary to myeloproliferative neoplasms (MPN-AML) cell lines) to ABT263, DT2216 (a BCL-XL selective PROTAC) (Khan et.al Nat. In summary, the novel BCL-XL/2 PROTAC 753B potently reduced cell viability in leukemia cell lines with diverse genetic background, in primary AML cells including venetoclax-resistant AML blasts, and extended survival of mice harboring human AML PDX in vivo without hematologic toxicity. Our data indicate that co-targeting of BCL-2 and BCL-XL may eliminate senescent AML cells surviving Ara-C chemotherapy, supporting the utility of future combination studies in leukemia.

We further investigated the molecular mechanism and showed that shRNA mediated knock-down or inhibition of BCL-XL, by either Navitoclax or the BCL-XL specific proteasomal degrader DT2216, results in significant induction of apoptosis in our models of AMKL...Furthermore, a genotype matched leukemic model of NUP98-KDM5A, which was presenting as monocytic (CD68+LYZ+) AML instead of AMKL, was resistant to either treatment with Venetoclax or Navitoclax whereas ML2, a MLL rearranged AML cell line, showed increased sensitivity to both Venetoclax and Navitoclax treatment...In conclusion, we generated a model of rare CG2 pediatric high-fatality leukemia that faithfully mimics the patient situation and allows biomass generation for large scale multi-omic approaches. Furthermore, we demonstrate a lineage- and genotype specific targetable dependency of pediatric AMKL towards inhibition of BCL-XL but not BCL2, that could be transferred to the clinic as a novel therapeutic option in high-risk infant leukemia.

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.

To overcome this toxicity, we treated FLC models with DT2216, a proteolysis targeting chimera (PROTAC) that directs BCL-XL for degradation via the von Hippel-Lindau (VHL) E3 ligase, which is minimally expressed in platelets. The combination of irinotecan and DT2216 in vitro on cells directly acquired from patients or in vivo using several xenografts derived from patients with FLC demonstrated remarkable synergy and at clinically achievable doses not associated with significant thrombocytopenia.

Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Collectively, our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore, its combination with a pro-apoptotic agent, i.e., DT2216, shows synergistic responses and can potentially overcome resistance.

Unlike other preclinical studies, like ABT263, DT2216 does not cause thrombocytopenia because VHL is poorly expressed in platelets. Our data suggests that the IC50 for all the T-ALL cell lines ranges from 2-100 nM, with the exception of SUP-T1, which exhibited an IC50 above 1 µM. Our data suggests that DT2216 could serve as a viable, safe, and potent alternative or addition to traditional clinical treatments of T-ALL.

Our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore its combination with a pro-apoptotic agent DT2216 shows synergistic responses and can potentially overcome resistance.

FLC is driven by a fusion oncokinase, DNAJB1-PRKACA, that arises from a deletion fusing exon 1 of DNAJB1 and exons 2-10 of PRKACA, the catalytic subunit of protein kinase A. Expression of this chimeric oncokinase is capable of recapitulating the key histologic and transcriptomic features of FLC...Unfortunately, Navitoclax has an on-target and dose-limiting toxicity of thrombocytopenia, which limits its clinical application...In conclusion, TOPO1 and Bcl-xL prove to be promising targets of interest in FLC. Targeting both with DT2216 and irinotecan leads to tumor shrinkage in pre-clinical models and offer a potential therapeutic/pharmacological intervention for FLC.

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.

We first evaluated the sensitivity of genetically diverse 17 leukemia cell lines, including 10 AML, 5 T-ALL and 2 MPN-AML to BCL-XL/BCL-2 dual inhibitor ABT263, 1 st generation BCL-XL PROTAC DT2216 ( Khan et al., Nature Medicine 2019 ) and BCL-XL/BCL-2 PROTAC 753B...We found that Ara-C indeed induced cellular senescence (SnCs) in MOLM-14 and Kasumi-1 AML cells, as manifested by increased cell size, induction of senescence-associated β-galactosidase activity (Fig...753B induced 50% cell killing at concentration of 1.3 μM, and nearly complete cell killing when combined with 5nM S63845 in OCI-AML2 at 24hr (Fig.1H), suggesting a synergistic effect in inducing apoptosis...753B showed potency comparable to ABT-263 in 15 AML samples, including 9 Venetoclax-resistant samples defined as IC50>1 μM (median IC 50 , 753B - 0.197 μM; ABT-263 - 0.280 μM) (Fig...In summary, BCL-XL/BCL-2 PROTAC 753B potently reduced cell viability through induction of apoptosis, and eliminated chemotherapy-induced senescent leukemia cells. In vivo efficacy studies of 753B combined with chemotherapy in the cell line- and patient-derived xenografts are ongoing and will be updated.

Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by GEM. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of GEM and their combination could be more effective for pancreatic cancer treatment.

P1, N=24, Recruiting, Dialectic Therapeutics, Inc | Not yet recruiting --> Recruiting | Trial completion date: Apr 2030 --> Apr 2023 | Initiation date: May 2021 --> Aug 2021