Dose-limiting Thrombocytopenia Research Articles

Session 5: Protein Degraders.

The so-called undruggable space is an exciting area of potential growth for drug development. Undruggable proteins are defined as those unable to be targeted via conventional small molecule drugs. New modalities are being developed to potentially target these proteins. Targeted protein degradation (TPD) is one such new modality, which over the last two decades has moved from academia to industry. TPD makes use of the endogenous degradation machinery present in all cells, in which E3 ubiquitin ligases mark proteins for degradation via ubiquitin attachment. This session explored the challenges and perspectives of using protein degraders as novel therapeutic agents. The session began with a general introduction to the modality, followed by considerations in evaluating their on- and off-target toxicities including data from an IQ Consortium working group survey. Unique absorption, distribution, metabolism, and excretion (ADME) properties of degrader molecules were presented in relation to their effect on drug development and nonclinical safety assessment. The role of transgenic models in evaluating hemotoxicity associated with cereblon-based therapies was then discussed. A case study to derisk dose-limiting thrombocytopenia was also presented. Finally, a regulatory perspective on the challenges of having toxicity associated with protein degraders was presented.

Abstract C052: Induction of senescence by paclitaxel in high-grade serous ovarian carcinoma (HGSOC) cells renders the cells resistant to paclitaxel but sensitizes them to BCL-xL targeting agents

Abstract High-grade serous ovarian carcinoma (HGSOC) is a very aggressive form of ovarian cancer, contributing to most ovarian cancer deaths. Paclitaxel is one of the major components of the first line treatment for HGSOC, but the development of chemoresistance to paclitaxel leads to the frequent relapse of the disease and poor prognosis in HGSOC patients. However, the mechanism underlying the chemoresistance has not been well established nor have the strategies to overcome the resistance been developed, which was investigated in the present study using three representative HGSOC cell lines (i.e. Kuramochi, OVCAR4, and OVCAR3 cells) in vitro. The results from our study show that induction of senescence contributes to paclitaxel-induced chemoresistance in HGSOC cells. More importantly, we found that senescent HGSOC cells induced by paclitaxel became highly dependent on BCL-xL for survival and thus were very sensitive to BCL-xL targeting agents, particularly to DT2216 (a BCL-xL specific proteolysis targeting chimera or PROTAC) and 753b (a BCL-xL and BCL-2 dual PROTAC). Because these PROTACs are more potent against senescent HGSOC cells but less toxic to platelets than ABT263 (a BCL-xL and BCL-2 dual inhibitor), they have the potential to be developed as new senolytics to overcome HGSOC chemoresistance to paclitaxel without causing the on-target and dose-limiting thrombocytopenia associated with ABT263 treatment. Citation Format: Yang Yang. Induction of senescence by paclitaxel in high-grade serous ovarian carcinoma (HGSOC) cells renders the cells resistant to paclitaxel but sensitizes them to BCL-xL targeting agents [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C052.

BCL-XL-targeting antibody-drug conjugates are active in preclinical models and mitigate on-mechanism toxicity of small-molecule inhibitors.

Overexpression of the antiapoptotic protein B-cell lymphoma-extra large (BCL-XL) is associated with drug resistance and disease progression in numerous cancers. The compelling nature of this protein as a therapeutic target prompted efforts to develop selective small-molecule BCL-XL inhibitors. Although efficacious in preclinical models, we report herein that selective BCL-XL inhibitors cause severe mechanism-based cardiovascular toxicity in higher preclinical species. To overcome this liability, antibody-drug conjugates were constructed using altered BCL-XL-targeting warheads, unique linker technologies, and therapeutic antibodies. The epidermal growth factor receptor-targeting antibody-drug conjugate AM1-15 inhibited growth of tumor xenografts and did not cause cardiovascular toxicity nor dose-limiting thrombocytopenia in monkeys. While an unprecedented BCL-XL-mediated toxicity was uncovered in monkey kidneys upon repeat dosing of AM1-15, this toxicity was mitigated via further drug-linker modification to afford AM1-AAA (AM1-25). The AAA drug-linker has since been incorporated into mirzotamab clezutoclax, the first selective BCL-XL-targeting agent to enter human clinical trials.

PROTAC-Mediated Dual Degradation of BCL-xL and BCL-2 Is a Highly Effective Therapeutic Strategy in Small-Cell Lung Cancer.

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. 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. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216 + venetoclax in reducing the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b was found to lead to significant tumor growth delay, similar to the DT2216 + venetoclax combination in H146 xenografts, by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. 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.

Safety and Tolerability of AZD0466 As Monotherapy for Patients with Advanced Hematologic Malignancies. Results from a Phase I/II Trial

Background: Overexpressionof B-cell lymphoma 2 (BCL-2)-related pro-survival proteins allows cancer cells to evade apoptosis, and targeting these proteins has become important in anticancer therapy. BCL-2 inhibition by venetoclax (VEN) is beneficial in many patients (pts) with acute myeloid leukemia (AML), but resistance is common due to upregulation of other pro-survival proteins such as BCL-extra-large (BCL-xL) and myeloid cell leukemia-1. Dual inhibition of BCL-2/xL has potential for broader activity than agents such as VEN. However, BCL-xL is critical for platelet survival and inhibition can cause dose-limiting thrombocytopenia (Konopleva and Letai. Blood 2018). To broaden therapeutic activity, we developed AZD0466, a drug-dendrimer conjugate in which the BCL-2/xL dual inhibitor AZD4320 is covalently conjugated to a pegylated poly-L-lysine dendrimer and gradually released by hydrolysis. Preclinically, AZD4320 showed greater inhibition of tumor growth than VEN and navitoclax in VEN-resistant xenograft models (Balachander et al. Clin Cancer Res 2020). A first-in-human study (NCT04214093) in pts with advanced solid tumors indicated that AZD0466 is well tolerated with no reported dose-limiting toxicities (DLTs). We present preliminary data from NIMBLE, a Phase I/II trial of AZD0466 in pts with advanced hematologic malignancies (NCT04865419). Methods: Module 1, Part A of NIMBLE is a dose-escalation study of AZD0466 monotherapy investigating its preliminary safety and tolerability, pharmacokinetics (PK), and preliminary efficacy. Dose escalation and de-escalation of AZD0466 follow a modified toxicity probability interval (mTPI-2) design. Eligible pts are ≥18 years old with relapsed/refractory (R/R) AML, acute lymphoblastic leukemia (ALL), or high-risk myelodysplastic syndrome. AZD0466 is administered IV with a dose ramp-up on Days (D) 1, 4, and 8, reaching the target dose on D8 of Cycle 1, with weekly administrations thereafter. Cycle duration is 35 days for Cycle 1 and 28 days for subsequent cycles. Pts remain on study until disease progression, unacceptable toxicity, or withdrawal of consent. Resul ts: As of May 31, 2023, 31 pts (n=25 AML, n=6 ALL; median age 69 years) had received ≥1 dose of AZD0466 at target doses of 300 mg (n=4), 600 mg (n=4), 1200 mg (n=7), 2400 mg (n=5), 3600 mg (n=9), and 5400 mg (n=2). Median treatment duration was 4.1 weeks (range 0.1−29.6). Twenty-two pts (71%) had prior VEN and 11 (35%) had prior hematopoietic stem cell transplantation. Treatment-emergent adverse events (TEAEs) of any grade reported in >10% of pts are displayed in Table 1. One Grade 5 intracranial hemorrhage unrelated to AZD0466 was reported. Four pts experienced a serious adverse event (SAE) possibly related to AZD0466 (alanine aminotransferase [ALT] increase, pneumonia, troponin T increase, and uncoded SAE). Grade ≥3 TEAEs possibly related to AZD0466, reported in 12 pts (39%), included febrile neutropenia (n=3), platelet count decrease (n=2), diarrhea, thrombocytopenia, ALT increase, blood creatinine phosphokinase increase, pneumonia, gamma-glutamyltransferase increase, lipase increased, and troponin T increase (n=1, each). DLTs of asymptomatic troponin increase were observed in two pts (3600 mg [n=1], 5400 mg [n=1]). Evaluation of maximum concentration and area under the concentration-time curve revealed dose-proportional exposure to AZD4320. Released AZD4320 represented 1-5% of total AZD4320 and had a longer half-life than total AZD4320. Preclinical modeling and PK data predicted that 5400 mg may be required to drive efficacy. As a surrogate of BCL-xL on-target activity, transient aggravation of thrombocytopenia post-dosing was observed across all dose cohorts followed by platelet recovery prior to the next dose. Platelet reduction was in line with preclinical models showing a substantial reduction at lower doses with a ceiling effect at higher doses. As of July 6, 2023, no complete responses have been observed. However, preliminary bone marrow (BM) blast reductions of >50% were observed in four pts (1200 mg [n=1], 2400 mg [n=2], 3600 mg [n=1]), indicating antileukemia activity of AZD0466. Conclusions: Given the observed DLTs of asymptomatic increase in troponin levels, and the lack of significant clinical benefit, the study was terminated.

Discovery of BCL-XL Heterobifunctional Degrader with Potentially Improved Therapeutic Window and Minimal Platelet Toxicity for Hematological Malignancies

Background In hematologic malignancies, apoptosis evasion promotes tumor formation/progression and is driven by the overexpression of anti-apoptotic proteins, including BCL-2 and BCL-XL. BCL-2-targeting small molecule inhibitor venetoclax won FDA approval for chronic lymphocytic leukemia and acute myeloid leukemia. Interests in developing BCL-XL-selective inhibitors arise as BCL-XL overexpression is implicated in venetoclax resistance. However, these efforts failed in clinical development due to dose-limiting thrombocytopenia, as platelets (PLT) also depend on BCL-XL for survival. To mitigate this on-target toxicity, a degrader strategy has been proposed, whereby BCL-XL protein can be selectively degraded by the von Hippel-Lindau protein (VHL) E3 ligase in tumor cells, but not in PLT which minimally express VHL. The first such BCL-XL degrader (DT2216) was developed and is now in early clinical trials. Despite negligible PLT toxicity in vitro, DT2216 was still toxic to PLT in vivo. 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. Warhead-containing metabolites could also form and behave as an inhibitor, exacerbating PLT toxicity in vivo. Utilizing our artificial intelligence-empowered neoDegrader platform, which accurately characterizes the landscape of the degrader-mediated, metastable interactions between E3 and its target protein, we were able to computationally design molecules that were relatively weak BCL-XL binders but could maintain the ternary complex at metastable states. Herein, we report the preclinical profile of the lead candidate NXD02. Methods We assessed the binding affinity of NXD02 to BCL-XL by surface plasmon resonance. We conducted flow cytometry to quantify NXD02-mediated BCL-XL protein degradation and apoptotic effects in MOLT4 cells. We also quantified the anti-proliferative activity of NXD02 by CellTiter-Glo assay. We measured protein level changes of apoptosis markers by western blot. We simultaneously determined plasma concentration of NXD02 and PLT at various time points following a single injection of NXD02 in rodents and monkeys. Finally, we evaluated the anti-tumor activity of NXD02 in an orthotopicMOLT4-luc mouse xenograft (CDX) model by bioluminescence imaging. Results The binding affinity of NXD02 to BCL-XL was > 40-fold weaker than that to DT2216 (K D 28.7 vs 0.6 nM). Despite this, NXD02 strongly degraded BCL-XL protein (DC 50 6.6 nM) with no obvious hook effect, which was more potent than DT2216 (DC 50 17.4 nM) in MOLT4 cells. At 100 nM, NXD02 markedly increased levels of active caspase 3 and cleaved PARP, which was consistent with evidence of massive apoptosis. This was translated to a potent anti-proliferative effect comparable to DT2216 (IC 50 27.8 vs 42.7 nM). NXD02 did not affect PLT viability up to a concentration of 10 μM in vitro. Notably, a single dose of NXD02 at 10 mg/kg i.v. achieved an exposure >4-fold than that of DT2216 (dose-normalized AUC 68.5 vs 15.3 μg*h/mL/(mg/kg)) in CD1 mice, while producing similar levels of peak PLT reduction (58% vs 65%) at ~24 h, which recovered by day 3. Comparable or less PLT reduction was also observed in rats (64% vs 66%) at 15 mg/kg and in cynomolgus monkeys (63% for NXD02 vs 87% for DT2216) at 3 mg/kg, all of which fully recovered by days 5-6, supporting the possibility for weekly dosing. Furthermore, NXD02 demonstrated stronger in vivo anti-tumor activity than DT2216 in a CDX model. Weekly i.v. dosing of NXD02 at 3 and 10 mg/kg achieved 80% and 100% tumor growth inhibition (TGI) by day 28 respectively, as compared to 36% TGI for DT2216 at 10 mg/kg. There was no significant body weight loss for all groups. Conclusions We have discovered a structurally-novel, VHL-mediated, heterobifunctional BCL-XL degrader NXD02 with “Weak Inhibitor, Strong Degrader” properties. NXD02 displayed, at the same dose, a much higher drug exposure, better anti-tumor activity and comparable platelet toxicity profile in vivo than the most advanced BCL-XL degrader DT2216. The results suggest that NXD02 may have a more favorable safety margin. 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.

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

BCL-XL Blockage with A1155463 Significantly Increases Efficacy of Venetoclax in Mantle Cell Lymphoma in Vitro and In Vivo

Mantle cell lymphoma (MCL) is a rare chronically relapsing subtype of aggressive B-cell non-Hodgkin lymphoma characterized by the canonical chromosomal translocation t (11;14) and other molecular cytogenetic aberrations including overexpression of BCL2 protein. Venetoclax (VEN), a BCL2 inhibitor, has demonstrated activity in MCL both as a monotherapy and in combination with other targeted agents. Treatment with single agent venetoclax is, however, hampered by frequent development of drug resistance caused in large part by adaptive upregulation of other anti-apoptotic BCL2 family members. Immunodeficient NOD-SCID-gamma mice were xenografted subcutaneously with MCL cell lines and PDX cells established from patients with relapsed and refractory MCL. The therapy was given for two weeks. VEN was administered by oral gavage (100 mg/kg/d), A1155463 was administered intraperitoneally (10 mg/kg/d). Western blotting was implemented to evaluate expression levels of BCL2 proteins, immunoprecipitation was used to analyze the levels of BIM bound to BCL2 and BCL-XL before and after exposure to VEN. CRISPR-Cas9 was employed to derive clones with BCL-XL knock-out. Blood cell counts in mice on A1155463 +/- VEN therapy were analyzed using the Mindray BC-5300 Auto Hematology Analyzer. In this study, we analyzed molecular mechanisms of in vivo acquired resistance to VEN using a panel of several murine cell line-based xenografts (CDX) and patient-derived xenografts (PDX) of MCL. First, the mice xenotransplanted with VEN-sensitive MCL cells were subject to monotherapy with VEN until development of resistant tumors (VEN-R). Western blot analysis of VEN-R tumors revealed upregulation of BCL-XL in majority of VEN-R tumors compared to controls. Other changes included upregulation of MCL1 and downregulation of BIM proteins in several models. Immunoprecipitation experiments confirmed that BCL-XL indeed serves as a buffer for BIM released from BCL2 after exposure to VEN thereby blocking VEN-triggered apoptosis. Importantly, we demonstrated that the upregulation of BCL-XL caused not only VEN resistance, but also led to BCL-XL-specific pro-apoptotic priming of VEN-R lymphoma cells. In vitro, the combination of VEN and A1155463, a specific BCL-XL inhibitor, induced cytotoxic synergy on a panel of MCL cell lines and primary cells. CRISPR-Cas9-mediated BCL-XL knock-out resulted in marked sensitization to VEN-induced apoptosis in MCL cell lines. In vivo, pharmacological blockage of BCL-XL strongly increased sensitivity to VEN. Despite that A1155463 exerted limited anti-lymphoma activity as monotherapy on tested CDX and PDX models, its combination with VEN was synthetically lethal and exerted significantly enhanced anti-lymphoma activity. The efficacy of the VEN and A1155463 combination was highly effective even in mice bearing VEN-R tumors (tumors with acquired VEN resistance, Figure 1). It has been published that mechanisms responsible for BCL-XL upregulation include activation of NFkappaB signaling via CD40 and hypoxia. Upregulation of BCL-XL induced by these factors in turn caused VEN resistance in vivo. Indeed, we confirmed that upregulation of BCL-XL was observed already after engraftment of VEN-sensitive MCL cells in immunodeficient mice. MCL cells isolated ex vivo from the established CDX tumors were significantly more sensitive to BCL-XL inhibition compared to the corresponding in vitro growing cell lines. Our results thus confirmed increased BCL-XL dependence of MCL cells in vivo compared to in vitro. We suggest that these microenvironmental factors were also critical for the selection of VEN-resistant clones during the therapy with VEN. Historically, experimental therapy of patients with chronic lymphocytic leukemia with navitoclax, a combined inhibitor of BCL2 and BCL-XL, was rater disappointing. The plausible reasons included both low efficacy of navitoclax due to insufficient BCL2 inhibition, and a dose-limiting thrombocytopenia. We demonstrated that the thrombocytopenia associated with continued therapy with A1155463 could be managed by 4 days on / 3 days off treatment strategy, which cannot be applied in case of fixed dual BCL2/BCL-XL inhibitors like navitoclax, or AZD4320. In summary, the combined inhibition of BCL2 and BCL-XL with VEN and A1155463 is a highly effective experimental treatment strategy for R/R MCL with a potential translation to the clinical grounds. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A first-in-human phase 1 study of oral LOXO-338, a selective BCL2 inhibitor, in patients with advanced hematologic malignancies (trial in progress).

TPS7582 Background: B-cell lymphoma 2 (BCL2) is a key regulator of apoptosis and provides protection from cell death in many hematologic malignancies. LOXO-338 is an orally bioavailable small molecule inhibitor of BCL2, developed to achieve selectivity over BCL-xL and thus avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. In preclinical studies, LOXO-338 showed a favorable pharmacological profile, selectively inhibited BCL2, and was well-tolerated in vivo. LOXO-338 also demonstrated dose-dependent tumor growth inhibition in various murine xenograft models and showed improved efficacy in combination with pirtobrutinib, a highly selective, non-covalent (reversible) BTK inhibitor (Brandhuber et al. Cancer Res 2021 81 (13 Suppl) 1258). Methods: LOXO-BCL-20001 is a global open-label, first-in-human phase 1 study of oral LOXO-338 in patients (pts) with advanced hematologic malignancies who have received prior therapy. The study will be conducted in 2 parts. Part 1 will evaluate LOXO-338 as monotherapy and will explore different dosing strategies. Part 2 will evaluate LOXO-338 in combination with pirtobrutinib. Part 1 dose escalation will follow an i3+3 design. Each cycle will be 28 days. Eligible pts include those with CLL/SLL, mantle cell lymphoma, and Waldenström macroglobulinemia (WM) who received standard therapy. Pts with other B-cell non-Hodgkin lymphomas (NHL) who received standard therapy, are not candidates for available therapy, or have no options with proven benefit are also eligible. Pts with active or suspected Richter transformation, transformed low grade lymphoma, Burkitt or Burkitt-like lymphoma, and multiple myeloma are also eligible. Pts with AL amyloidosis are eligible in monotherapy dose expansion; all other pts are eligible in both monotherapy and combination dose expansion. Pts must not have progressed while receiving prior BCL2 inhibitor; those with WM or AL amyloidosis must not have received a prior BCL2 inhibitor. Key exclusion criteria include history of CNS involvement, stem cell transplant or CAR-T therapy <60 days, concurrent anticancer therapy, and clinically significant cardiovascular disease. Primary objective is to determine the recommended phase 2 dose of oral LOXO-338 in pts who were previously treated for CLL/SLL and other B-cell NHL, administered alone or in combination with pirtobrutinib. Determining anti-tumor activity in pts with WM and AL amyloidosis is an additional primary objective of part 1 monotherapy. Secondary objectives include determining the safety profile, PK, and preliminary efficacy of LOXO-338 administered alone and in combination with pirtobrutinib. Antitumor activity will be evaluated based on overall response rate (ORR), progression-free survival (PFS), time to progression (TTP) and duration of response (DOR) according to disease-specific response criteria. Clinical trial information: NCT05024045.

A phase II study of Navitoclax (ABT-263) as single agent in women heavily pretreated for recurrent epithelial ovarian cancer: The MONAVI – GINECO study

BackgroundThere are limited treatment options for ovarian cancer patients with early relapse after platinum chemotherapy. In preclinical studies, we previously demonstrated the promising activity of ABT-737, a Bcl-2/Bcl-xL anti-apoptotic protein inhibitor, in chemo-resistant ovarian cancer cells and tumors, suggesting its potential activity in platinum-resistant patients. MethodsWe conducted a prospective multicenter single-arm phase II study to assess the efficacy of Navitoclax (orally available ABT-737 analogue) monotherapy in 46 heavily pretreated (2–12 lines, median = 4) patients with high-grade serous platinum-resistant ovarian tumors. Navitoclax was administered at the daily dose of 150 mg during a lead-in period (7–14 days) and then increased to 250 mg daily in the absence of dose-limiting thrombocytopenia (<G3). Progression-free survival (PFS) based on RECIST v1.1 criteria was the primary endpoint. Analysis of efficacy according to the expression of Bcl-2 family proteins in tumor biopsies was also planned. ResultsThe 3-month PFS was 22.7% [95%CI: 13.2–39.2], median PFS was 1.64 months [95%CI: 1.58–2.30]. There were 16 (35.6%, 95%CI: 22.3–51.3) overall responses (RECIST v1.1): 1 partial response and 15 stable diseases. No correlation between the expression of Bim, Mcl-1 and P-ERK with clinical response was found in this study.Thrombocytopenia was the major side-effect (G3/4: n = 12; 26%), leading to pursue at the daily dose of 150 mg in 8 patients and to discontinue treatment in 3 patients. Neither significant bleeding nor toxic death were observed. ConclusionsNavitoclax monotherapy had poor activity that was not correlated with the expression of Bim, Mcl-1 and P-ERK, without unacceptable toxicity.Trial Registration: Clinicaltrials.gov identifier: NCT02591095

A First-in-Human Phase 1 Study of Oral LOXO-338, a Selective BCL2 Inhibitor, in Patients with Advanced Hematologic Malignancies (Trial in Progress)

Background: B-cell lymphoma 2 (BCL2) is a key regulator of apoptosis and provides protection from cell death in many hematological malignancies. The BCL2 inhibitor venetoclax is approved for the treatment of CLL/SLL and acute myeloid leukemia and has activity in other lymphoid malignancies. LOXO-338 is a novel, orally bioavailable small molecule inhibitor of BCL2, designed to achieve selectivity over BCL-xL and thus avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. In preclinical studies, LOXO-338 showed a favorable pharmacological profile, selectively inhibited BCL2, and was well-tolerated in vivo. LOXO-338 also demonstrated dose-dependent tumor growth inhibition in various murine xenograft models, and showed improved efficacy in combination with pirtobrutinib, a highly selective, non-covalent BTK inhibitor (Brandhuber et al. Cancer Res 2021; 81, 13 Supplement, 1258).Study Design and Methods: LOXO-BCL-20001 is an open-label, multi-center, first-in-human Phase 1 study of oral LOXO-338 in patients with advanced hematologic malignancies who have received standard therapy. The study will be conducted in 2 parts. Part 1 will evaluate LOXO-338 as monotherapy, and will explore different dosing strategies. Part 2 will evaluate LOXO-338 in combination with pirtobrutinib. The dose escalation portion of the study in Part 1 will follow an i3+3 design. Each cycle will be 28 days (4 weeks).Eligible patients include those with CLL/SLL, mantle cell lymphoma (MCL), and Waldenstrӧm macroglobulinemia (WM) who have already received standard therapy. Patients with other B-cell non-Hodgkin lymphomas (NHLs) who failed standard therapy or, in the opinion of the investigator, have no known available options to provide benefit for the patient's condition, are also eligible. Patients must have recovered from prior treatment-related adverse events. Patients with active or suspected Richter transformation, transformed low grade lymphoma, Burkitt or Burkitt-like lymphoma, and multiple myeloma (MM) are eligible in dose-expansion. Key exclusion criteria include history of CNS involvement, stem cell transplant or CAR-T therapy <60 days, concurrent anticancer therapy, and clinically significant cardiovascular disease.The primary objective of Part 1 is to determine the maximum tolerated dose (MTD)/ recommended Phase 2 dose (RP2D) of oral LOXO-338 in patients who were previously treated for CLL/SLL and other B-cell NHLs. Key secondary objectives include determining the safety and tolerability, and pharmacokinetic properties of LOXO-338. Antitumor activity will be evaluated based on overall response rate (ORR), progression-free survival (PFS), time to progression (TTP) and duration of response (DOR) based on disease-specific response criteria per investigator assessment. Key objectives of part 2 are to determine the safety profile and tolerability, PK properties, and anti-tumor activity of LOXO-338 in combination with pirtobrutinib. DisclosuresAlencar: Seattle Genetics: Consultancy; Kite Pharma: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Epizyme: Consultancy; Celgene: Consultancy; BeiGene: Consultancy; Amgen: Consultancy. Roeker: AbbVie, AstraZeneca, Janssen, LOXO, Pharmacyclics, TG Therapeutics, Vaniam Group, Verastem: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy, Research Funding; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Abbot Laboratories: Current equity holder in publicly-traded company. Hoffmann: TG Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pharmcyclics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria. Guru Murthy: Cancerexpertnow: Honoraria; Guidepoint: Consultancy; Techspert: Consultancy; Qessential: Consultancy; Cardinal Health Inc.: Honoraria; TG therapeutics: Other: Advisory board. Patel: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Ku: Loxo Oncology at Lilly: Current Employment, Current holder of stock options in a privately-held company. Pauff: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Eyre: Incyte: Consultancy; Loxo Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Gilead/KITE: Honoraria, Other: Travel support for conferences, Research Funding, Speakers Bureau; Secura Bio: Consultancy, Honoraria; Janssen: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel to conferences; AstraZeneca: Honoraria, Research Funding. Jurczak: Celtrion: Research Funding; Celgene: Research Funding; Debbiopharm: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Sandoz: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Epizyme: Research Funding; Incyte: Research Funding; Merck: Research Funding; Takeda: Research Funding; TG Therapeutics: Research Funding. OffLabel Disclosure:LOXO-338 is a novel, orally bioavailable small molecule inhibitor of BCL2 for advanced hematologic malignancies.

Abstract 1145: AGX323 - A SAM-competitive, orally available inhibitor of protein arginine methyltransferase 5 (PRMT5) with potent cellular antiproliferative and in vivo antitumor activity against selected solid cancer types

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is an attractive therapeutic target because of its involvement in key oncogenic mechanisms including epigenetics, DNA response and RNA processing. Early development of PRMT5 inhibitors has focused on hematological malignancies although more recently several compounds have entered the clinic for both liquid and solid malignancies. Early Phase I clinical data indicate dose-limiting hematological toxicity, particularly anemia and thrombocytopenia. We focused our effort in discovering compounds with potent inhibitory activity against cells from solid cancer types known to overexpress PRMT5 and which correlated with poor prognosis (NSCLC, breast, bladder and pancreatic cancers). Methods: Enzyme inhibition was conducted using a PRMT5:MEP50 flash plate assay, with S-adenosylmethionine (SAM) as the methyl donor and histone H4 as substrate. Inhibition of cell proliferation was assessed in various cancer lines using a 7-day cellTitre-Glo (CTG) assay. Binding mode of the inhibitors to PRMT5 was investigated using surface plasmon resonance (SPR). In vivo antitumor activity was evaluated in various human cancer models grown in immunocompromised mice. Results: Structure activity relationship of a series of over 50 close analogs demonstrated that in vitro anticancer activities correlate poorly with enzyme inhibition potency. To understand this discrepancy we used SPR to study the PRMT5 binding mode of cell active and inactive compounds in the presence of various endogenous substrates (SAM, SAH or MTA). We obtained binding affinity (KD), association constant (Ka) and dissociation constant (Kd) of exemplars of active and inactive inhibitors and correlated their relationship to cell activity. The lead compound AGX323 has a KD of 1.8 x 10-10 M to SAM-bound PRMT5 and exhibited potent antiproliferative activity against a panel of over 20 human solid epithelial cancer cell lines from lung, breast, bladder and pancreas, with nM potency. For example, in the H1048 human lung cancer model it has an enzyme inhibition IC50 of 0.8 nM, a cell proliferation IC50 of 1.7 nM and when given to tumor bearing mice it demonstrated robust antitumor activity at well-tolerated dose levels. In nonclinical ADME studies, AGX323 demonstrated favorable drug-like properties: including acceptable oral bioavailability (in mouse, dog and monkey) and systemic exposure following oral administration, excellent metabolic stability, lack of significant drug-drug interaction liabilities (transporters, CYP inhibition, CYP induction). Conclusion: AGX323 demonstrated potentially best-in-class activity in PRMT5 dependent human solid cancer models and exhibited excellent drug-like properties. IND enabling studies are currently planned. Citation Format: Francis Y. F. Lee, Wen-Lian Wu, Zhiqiang Yang, John Tan. AGX323 - A SAM-competitive, orally available inhibitor of protein arginine methyltransferase 5 (PRMT5) with potent cellular antiproliferative and in vivo antitumor activity against selected solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1145.

Abstract 1258: Preclinical characterization of LOXO-338, a novel, oral and selective BCL2 inhibitor

Abstract BCL2 is an important therapeutic target in various hematologic malignancies. Venetoclax is the only FDA-approved BCL2 inhibitor, indicated for use in CLL/SLL and acute myeloid leukemia (AML). LOXO-338 is a novel, oral BCL2 inhibitor, designed to achieve selectivity over BCL-xL (Lin S, et al. AACR, 2019), to avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. Here, we present updated LOXO-338 preclinical characterization of its potency in biophysical and cellular assays, selectivity for BCL2 over other BCL2 family members, and efficacy in murine tumor growth models to support its advancement into human clinical trials. In homogeneous time resolved fluorescence (HTRF) assays measuring the molecular interactions of BCL2 and other BCL2 family members to BAK, LOXO-338 preferentially inhibited the BCL2/BAK interaction with an IC50 value of 4.5 nM; whereas the interactions between BCLxL/BAK, and MCL1/BAK were inhibited by LOXO-338 with IC50 values of 49.6 nM and &amp;gt;10,000 nM, respectively. In cellular immunoprecipitation assays, LOXO-338 showed dose-dependent inhibition of BCL2/BIM interactions in RS4;11 cells (acute lymphoblastic leukemia (ALL) cell line) at concentrations ranging from 30-3000 nM and no inhibition of MCL-1/NOXA interactions in Karpas 299 cells (anaplastic large cell lymphoma cell line) at 3000 nM, the highest concentration tested. Using high-content imaging to measure the effects of LOXO-338 on cellular proliferation we determined LOXO-338 inhibited proliferation of RS4;11 cells with an IC50 value of 2 nM, whereas the IC50 values for the BCL2 independent cell lines H146 (small cell lung cancer (SCLC) cell line), Daudi (Burkitt's lymphoma cell line), and KMS11 (multiple myeloma cell line) cells were greater than 2000 nM. Anti-proliferative effects of LOXO-338 on a panel of cancer cell lines derived from different tissues were assessed using CCK-8 assays. LOXO-338 inhibited the proliferation of cell lines derived from follicular lymphoma (FL), diffuse large B-cell lymphoma, AML, and ALL with IC50 values ranging from 7 nM to 78 nM. In murine tumor xenograft models using FL, AML, and ALL cell lines, LOXO-338 administered once daily demonstrated dose-dependent tumor growth inhibition. LOXO-338 was well-tolerated by the tumor-bearing mice and no significant weight loss of the mice was observed in the studies. The antitumor potency of LOXO-338 is similar to comparable doses of venetoclax in these models. Novel preclinical combination data and comparative profiles to venetoclax will be presented. This preclinical profile of LOXO-338 supports its nomination as a novel BCL2 inhibitor clinical candidate. A first-in-human Phase 1 clinical trial is planned for 2021. Citation Format: Barbara Brandhuber, Karin Ku, Maria J. Lallena, Carmen Baquero, Regina Choy, Kevin Ebata, Sophie Shu Lin, Lihua Jiang, Yanxin Liu, Xiangling Chen, Liguang Lou. Preclinical characterization of LOXO-338, a novel, oral and selective BCL2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1258.

Pharmacokinetic\u2013pharmacodynamic guided optimisation of dose and schedule of CGM097, an HDM2 inhibitor, in preclinical and clinical studies

BackgroundCGM097 inhibits the p53-HDM2 interaction leading to downstream p53 activation. Preclinical in vivo studies support clinical exploration while providing preliminary evidence for dosing regimens. This first-in-human phase I study aimed at assessing the safety, MTD, PK/PD and preliminary antitumor activity of CGM097 in advanced solid tumour patients (NCT01760525).MethodsFifty-one patients received oral treatment with CGM097 10–400 mg 3qw (n = 31) or 300–700 mg 3qw 2 weeks on/1 week off (n = 20). Choice of dose regimen was guided by PD biomarkers, and quantitative models describing the effect of CGM097 on circulating platelet and PD kinetics.ResultsNo dose-limiting toxicities were reported in any regimens. The most common treatment-related grade 3/4 AEs were haematologic events. PK/PD models well described the time course of platelet and serum GDF-15 changes, providing a tool to predict response to CGM097 for dose-limiting thrombocytopenia and GDF-15 biomarker. The disease control rate was 39%, including one partial response and 19 patients in stable disease. Twenty patients had a cumulative treatment duration of >16 weeks, with eight patients on treatment for >32 weeks. The MTD was not determined.ConclusionsDespite delayed-onset thrombocytopenia frequently observed, the tolerability of CGM097 appears manageable. This study provided insights on dosing optimisation for next-generation HDM2 inhibitors.Translational relevanceHaematologic toxicity with delayed thrombocytopenia is a well-known on-target effect of HDM2 inhibitors. Here we have developed a PK/PD guided approach to optimise the dose and schedule of CGM097, a novel HDM2 inhibitor, using exposure, platelets and GDF-15, a known p53 downstream target to predict patients at higher risk to develop thrombocytopenia. While CGM097 had shown limited activity, with disease control rate of 39% and only one patient in partial response, the preliminary data from the first-in-human escalation study together with the PK/PD modeling provide important insights on how to optimize dosing of next generation HDM2 inhibitors to mitigate hematologic toxicity.

AZD4320, A Dual Inhibitor of Bcl-2 and Bcl-xL, Induces Tumor Regression in Hematologic Cancer Models without Dose-limiting Thrombocytopenia.

Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL-mediated thrombocytopenia. We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2- and Bcl-xL-dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL-dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

Venetoclax and Navitoclax in Pediatric Patients with Acute Lymphoblastic Leukemia and Lymphoblastic Lymphoma

Venetoclax and Navitoclax in Pediatric Patients with Acute Lymphoblastic Leukemia and Lymphoblastic Lymphoma

Abstract CT153: SDX-7320 elicits improvements in tumor-related and metabolic biomarkers: Results of a phase 1 dose-escalation study in patients with advanced refractory or late-stage solid tumors

Abstract Background and Rationale: Metabo-oncology is the study of specific tumor types whose growth and/or metastasis is accelerated by metabolic dysfunction, often associated with hyper-adiposity. In this setting the metabolic hormones leptin, adiponectin and insulin are potential mediators. SDX-7320 is a novel methionine aminopeptidase type 2 inhibitor (MetAP2) being developed for use in combination with other anti-cancer agents to treat the metabo-oncology patient population. Study Design: This first-in-human study used a 3+3 design with dose expansion at the RP2D. Measurements included standard assessments of safety, tolerability, as well as inhibition of MetAP2 in whole blood, plasma levels of protein biomarkers and drug exposure. Tumor response was measured using RECIST v.1.1. Results: 32 patients with an average of 5.3 prior treatment regimens were dosed. Sub-cutaneous dosing of SDX-7320 began at 1.7 mg/m2 once per week (Q7D, 28 days/cycle) and was escalated to 49 mg/m2. Repeated Q7D administration at 49 mg/m2 resulted in dose-limiting (G3-G4) thrombocytopenia. Expansion at 49 mg/m2 confirmed thrombocytopenia as the dose-limiting toxicity (DLT), reversible upon cessation of dosing. Dose-escalation was re-initiated at 36 mg/m2 every two weeks (Q14D). Repeated dosing at 65 mg/m2 (Q14D) resulted in G3 reversible thrombocytopenia. Expansion at 49 mg/m2 (Q14D) confirmed that this was the maximum tolerated dose (MTD) on a Q14D schedule. No deaths or Grade 5 TEAEs attributable to SDX-7320 were reported. Six patients had a combined total of 9 Grade 3/4 TEAEs considered by the Investigator to be at least possibly SDX-7320-related: thrombocytopenia (4 patients) and single cases of anemia, anorexia, fatigue, QTc prolonged, and vasculitis. Fourteen patients (44% had stable disease &amp;gt2 cycles and three patients had stable disease for ≥6 cycles. Inhibition of MetAP2 in whole blood was 100% at all doses evaluated while the time to reach 100% inhibition was inversely related to dose. When stratified for elevated baseline levels, the following biomarkers declined after initiating SDX-7320 (average of each patient's maximum % change relative to baseline +/- SD): bFGF (-53 +/- 76%), VEGF-C (-35 +/- 39%), and insulin (-55 +/- 30%). Without stratification, leptin levels declined (-51 +/-29%) and levels of adiponectin increased after SDX-7320 (+74 +/- 63% relative to baseline). Conclusions and Next Steps : SDX-7320 was well-tolerated with prolonged stable disease in an open-label, phase I first-in-human study. Improvements were observed in both tumor-related and metabolic biomarkers. Additional studies of SDX-7320 (in combination with other anti-cancer agents) in patients with solid tumors sensitive to metabolic hormones are scheduled. SDX-7320 is the first novel agent in clinical development to treat the metabo-oncology patient population. Citation Format: Monica M. Mita, Johanna Bendell, Alain C. Mita, Michael Gordon, Jasgit Sachdev, Bradley J. Carver, James Shanahan, Benjamin Mayes, Kris Awerkamp, David Browning, Neal Salomon, Kimberly Sullivan, Alfred Anderson-Villaluz, Joe Johnson, John S. Petersen, David J. Turnquist, Peter Cornelius. SDX-7320 elicits improvements in tumor-related and metabolic biomarkers: Results of a phase 1 dose-escalation study in patients with advanced refractory or late-stage solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT153.

A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity.

BCL-XL is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors such as ABT263 as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but significantly less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing significant thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.

DT2216, a BCL-XL Proteolysis Targeting Chimera (PROTAC), Is a Potent Anti T-Cell Lymphoma Agent That Does Not Induce Significant Thrombocytopenia

There is an urgent need for new therapeutic agents to treat patients with T-cell lymphoma (TCL). Multiple hematologic malignancies evade apoptosis through overexpression of anti-apoptotic proteins in the BCL-2 family, including BCL-2, BCL-XL, and MCL-1. We and others recently showed that a large fraction of cutaneous and peripheral TCL cell lines, patient-derived xenografts and primary patient samples depend on BCL-XL for survival (Koch et al, Blood. 2019; 133:566-575). These findings suggest that targeted inhibition of BCL-XL could offer therapeutic benefit for some TCL patients. Currently available small molecule BCL-XL inhibitors have failed during clinical development due to on-target and dose-limiting thrombocytopenia, as platelets depend on BCL-XL for survival. To overcome this toxicity, we developed DT2216, a novel proteolysis-targeting chimera (PROTAC) that targets BCL-XL to the Von Hippel Lindau (VHL) E3 ligase for proteasomal degradation. We selected the VHL ligase because platelets express very low levels of VHL, suggesting that they would be spared from the pro-apoptotic effects of DT2216. Here we examined the therapeutic potential of DT2216 against different TCL cell lines in vitro and in TCL xenograft mouse models. We first profiled the expression of different anti-apoptotic BCL-2 family proteins in multiple TCL cell lines (Fig. 1a) and tested their sensitivity to selective inhibitors for different BCL-2 family proteins as well as commonly-used chemotherapeutic agents (Table 1). The results showed that the TCL cells with higher levels of BCL-XL were more resistant to doxorubicin, etoposide and vincristine. DT2216 was more effective in reducing the viability of BCL-XL-dependent TCLs such as MyLa cells than ABT263 (a dual BCL-2/XL inhibitor). The EC50 value of DT2216 for MyLa TCL cells was less than 10 nM and DT2216 killed MyLa cells through induction of BCL-XL degradation and cellular apoptosis (Fig. 1b-f). Moreover, DT2216 was less toxic to human platelets than ABT263 in vitro with an EC50 &amp;gt; 3 μM. We further validated the effect and specificity of DT2216 in MJ cells, another BCL-XL-dependent TCL cell line, and demonstrated that its antitumor activity was dependent on proteasome activity. In vivo, when DT2216 was given to mice with MyLa TCL xenografts by i.p. injections at 10 mpk/q4d, it significantly inhibited tumor growth, whereas ABT263 at the same dose had no significant effect (Fig. 1g). More importantly, after MyLa xenografted mice failed to respond to ABT263 treatment, we subjected the mice to DT2216 (10 mpk/q4d), which induced rapid tumor regression and increased the survival of the mice without causing significant reduction of blood platelets (Fig. 1h). These effects were associated with a significant reduction in BCL-XL expression and activation of caspase cascade in tumor xenografts. However, some TCLs depend on not only BCL-XL but also BCL-2 or MCL-1 for evasion of apoptosis. Therefore, we next assessed the therapeutic potential of DT2216 in combination with a selective BCL-2 inhibitor using the TCL PDX DFTL-28776, which depends on both BCL-XL and BCL-2 for survival. We found that the combination of DT2216 with ABT199 (a selective BCL-2 inhibitor) could more effectively kill DFTL-28776 TCL cells than either agent alone in cell culture. The effect of the combination treatment on the growth of DFTL-28776 PDX in vivo is under examination. Collectively, our findings suggest that targeting BCL-XL using DT2216 can selectively kill BCL-XL-dependent TCL cells without causing significant platelet toxicity. Moreover, the combination of DT2216 with an inhibitor targeting other anti-apoptotic BCL-2 family proteins may have broad therapeutic utility against multiple TCL types and other cancers dependent on BCL-XL. Disclosures He: University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Khan:University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zhang:University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Weinstock:Celgene: Research Funding. Zhou:Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents.