Background Cell division cycle 7-related protein kinase (CDC7), or DBF4-dependent CDC7 kinase (DDK), is a cell cycle kinase that maintains DNA replication by phosphorylation and activation of the minichromosome maintenance protein 2 and 4 (MCMs), components of the replicative DNA helicase. Due to the central role of CDC7 in maintenance of the replication fork integrity, chemical inhibition of CDC7 kinase can ultimately lead to cancer cell death. SGR-2921 is an oral, small molecule inhibitor of CDC7. Preclinical studies demonstrate that, among all cell lines tested, SGR-2921 has the most potent anti-proliferative activity in AML. Potent antitumor activity has also been demonstrated in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) AML models. This antitumor response is observed in animal models representative of difficult to treat patient populations and appears to be agnostic of serious mutations (including those with p53 mutations and FLT3 mutations), BTK resistance, and multiple prior lines of treatment. Patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) or high-risk (HR) or very high-risk (VHR) myelodysplastic syndrome (MDS) have low response rates and poor overall survival. Considering the potent preclinical activity of SGR-2921 in AML and MDS models, coupled with the high unmet medical need in this population, SGR-2921 is being evaluated in patients with R/R AML and HR/VHR MDS. Study Design and Methods This is a phase 1, FIH, open-label, single-agent, two-arm, dose escalation study (NCT#05961839) designed to evaluate safety and tolerability and identify the recommended phase 2 dose (RP2D) of SGR-2921 as monotherapy in subjects with R/R AML, HR MDS, or VHR MDS. The study utilizes a hybrid accelerated titration design with single patient cohorts that transitions to a 3+3 design once a single Grade 2 event or DLT is observed. This is a multicenter global study (US, Spain and France) with an estimated study recruitment start date in October, 2023. SGR-2921 will be administered orally, once daily, utilizing a 5-day on and 9-day off dosing schedule over a 28-day cycle. Up to a maximum of 144 patients will be enrolled in the dose escalation and exploratory cohort phase of the study. To evaluate the effect of CYP3A4 inhibition on SGR-2921 exposure, subjects will be enrolled into one of two staggered, parallel study treatment arms, according to concomitant administration with (Arm B) or without (Arm A) azole antifungals that are strong CYP3A4 inhibitors at time of first dose. Safety and tolerability must be demonstrated in treatment Arm A, at the first two dose levels before initiating treatment Arm B. A single dose PK run-in will be required for subjects enrolled into the first 3 cohorts of each treatment arm. Subjects will be treated at increasing doses of study drug until all dose levels have been investigated or any dose level is found to exceed the maximum tolerated dose (MTD). A RP2D will be selected from one of the tolerable dose levels which will not exceed the MTD (see Figure 1 for study design). Key study inclusion criteria include: Age ≥ 18 years of age; Life expectancy ≥ 8 weeks; Confirmed diagnosis of R/R AML or HR and VHR MDS; Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2. Key study exclusion criteria include: Active malignancies not related to AML or MDS within two years prior to the first dose or requiring ongoing treatment; Clinical evidence of central nervous system (CNS) or pulmonary leukostasis; ≥ Grade 3 disseminated intravascular coagulation, or active CNS leukemia; QT interval corrected for heart rate per Fridericia's formula ≥470 msec during screening ECG. The study primary objectives are to evaluate the safety and tolerability of SGR-2921 as monotherapy and identify RP2D including MTD. Secondary objectives include evaluating the pharmacokinetics (PK) of SGR-2921 and investigating preliminary antitumor activity (composite complete remission rate, objective response rate, duration of response, etc.).

Introduction: Elevated replication stress (RS) is a common feature of many highly proliferative cancers including AML. CDC7 has been shown to activate key components of the RS response, including ATR, and the BRCA1 and cohesin complexes, thereby mitigating accumulation of DNA damage and genomic instability. Inhibition of CDC7 in cancer cells results in an impaired response to RS, leading to accumulation of DNA damage and cell death. SGR-2921 is a potent inhibitor of CDC7 and a clinical trial to evaluate the safety, tolerability and preliminary antitumor activity of SGR-2921 in patients with AML and MDS is expected to begin in 2023 (NCT 05961839). We have previously shown that SGR-2921 has potent anti-proliferative activity in 12 out of 16 patient-derived AML samples ex vivo. Importantly, 6 out of 6 AML patient samples with mutations in TP53 responded to SGR-2921. Here we show that SGR-2921 results in potent, dose-dependent, anti-leukemic activity in multiple disseminated in vivo AML patient-derived xenograft (PDX) models representing difficult-to-treat disease. Methods: NOG-EXL mice were inoculated with primary human AML blast cells after sublethal irradiation. Engraftment of human AML blast cells were measured in surrogate mice by flow cytometry using human CD45 vs. mouse CD45 markers. When human CD45 engraftment reached >20% of bone marrow cells in surrogate mice, the study mice were randomized based on weight and dosed with vehicle, SGR-2921 at 3 dose levels or a standard of care agent. SGR-2921 was administered via oral gavage (PO) twice daily in cycles of 5 days of dosing followed by 9 days of no compound dosing over 2 cycles. In a separate study, phosphorylated MCM2 (a direct substrate of CDC7) was measured in the same cohort of mice engrafted with human AML cells following 3 doses of SGR-2921 to determine target engagement. Results: In vivo, SGR-2921 showed strong anti-leukemic activity in the AML PDX models at tolerated doses. Specifically, engrafted AML cells derived from a patient with TP53 mutated AML, a relapsed and refractory patient and a patient with KTM2a rearrangements showed significant and dose-dependent reduction in human CD45 positive AML blast cells. The reduction of blast cells was observed in both peripheral blood and bone marrow. The reduction in human CD45 positive blast cells were associated with an increase in mouse CD45 positive cells. In addition, SGR-2921 demonstrated dose-dependent inhibition of phosphorylated MCM2 in the spinal cord and spleen of the AML PDX models as surrogates of the bone marrow and extravascular compartments, respectively. Conclusions: Our data show remarkable dose-dependent in vivo activity of SGR-2921 in AML PDX models, including in those representing difficult-to-treat disease. Direct inhibition of CDC7 by SGR-2921 in AML blasts was demonstrated by a dose-dependent reduction of phosphorylated MCM2. Together, these data demonstrate that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with potential utility in patients with high risk mutations and relapsed and refractory AML.

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Previously, we described the discovery of novel MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells and the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models as well as combination potential with agents including standard-of-care (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). When administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, SGR-1505 demonstrated tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential for patients with selected B-cell lymphomas. Here we further characterized SGR-1505, in a series of in vitro and ex vivo assays, as well as RNA-seq analysis to examine changes in gene expression from in vivo tumor samples. We also compared SGR-1505 with a competitor Phase I candidate, JNJ-67856633 (JNJ-6633) (ref 3, 4). Results: SGR1505 potency and downstream effects were evaluated in a series of biochemical and cell based assays. SGR-1505 showed excellent potency in the biochemical assay and strong anti-proliferative effects on ABC-DLBCL cells. SGR-1505 was more potent than JNJ-6633 in all assays tested (Table 1). These results are also consistent with the result from a human primary T-cell based assay, where SGR-1505 showed at least ten-fold better potency than JNJ-6633. RNA-seq analysis was conducted to examine changes in gene expression from in vivo tumor samples. Greater modulation of BIOCARTA NF-kB pathway genes was seen with SGR-1505 compared to JNJ-6633, as measured by mean absolute change in gene expression. At 6 hr and later timepoints, we also observed a trend of increases in genes related to cell cycle pathways, such as cell cycle, DNA damage, and apoptosis. SGR-1505 is being evaluated in the SGR-1505-102 phase 1 study, which is an ongoing first-in-human, single center, dose escalation study to evaluate the safety, tolerability, PK and PD of SGR-1505 tablets in healthy participants (ACTRN12623000358640p). Preliminary data showed changes in target engagement markers at concentrations predicted by the in vitro and ex vivo assays, consistent with MALT1 protease inhibition. Conclusions: SGR-1505, a MALT1 protease small molecule inhibitor, consistently demonstrated better potency in in vitro and ex vivo assays when compared to the clinical-stage JNJ-6633 compound and greater effects on NF-kB pathway gene expression in in vivo tumor samples based on RNA-seq analysis. Changes in biological pathways mediated by MALT1 were also observed at relevant doses in the ongoing SGR-1505 healthy volunteer study. Currently, a phase 1 clinical trial in patients with mature B cell neoplasms is also ongoing (NCT05544019). The data presented suggests SGR-1505 has a potential best-in-class profile and supports advancing the ongoing clinical development of SGR-1505.

MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of the nuclear factor kappa B (NF-κ B) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of the NF-κ B is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models.

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of the nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. In particular, constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity, and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. Aims: These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential and may expand therapeutic options for patients with selected B-cell lymphomas supporting further evaluation of SGR-1505 in clinical trials. Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies. At present, the study has been activated in 3 sites in the United States. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1–6 subjects, and at higher dose levels using a conventional 3+3 design. Encore Abstract—previously submitted to EHA 2023 The research was funded by: Schrodinger Keywords: aggressive B-cell non-Hodgkin lymphoma, molecular targeted therapies, ongoing trials Conflicts of interests pertinent to the abstract. B. Yoo Employment or leadership position: Schrodinger Stock ownership: Schrodinger Z. Nie Employment or leadership position: Schrodinger Stock ownership: Schrodinger G. Krilov Employment or leadership position: Schrodinger Stock ownership: Schrodinger J. Tan Employment or leadership position: Schrodinger Stock ownership: Schrodinger H. Wright Employment or leadership position: Schrodinger Stock ownership: Schrodinger D. Weiss Employment or leadership position: Schrodinger Stock ownership: Schrodinger W. Yin Employment or leadership position: Schrodinger Stock ownership: Schrodinger K. Akinsanya Employment or leadership position: Schrodinger Stock ownership: Schrodinger

Introduction: CDC7 is a protein kinase that is active during the S-phase of the cell cycle. CDC7 plays a critical role in the replication stress response by phosphorylation and activation of the BRCA1 and Cohesin complexes, which protect and restart stalled replication forks. In addition, by maintaining DNA helicase activity during replication fork stalling, CDC7 establishes a platform for ATR checkpoint activation. Acute myeloid leukemia (AML) is typically a rapidly proliferating cancer and is characterized by high replication stress and DNA damage. Agents that target replication stress and DNA damage response pathways represent novel therapeutic opportunities in AML. Here we show that inhibition of CDC7 in AML models results in potent anti-tumor activity by amplifying replication stress and DNA damage. We show that CDC7 inhibition has synergistic anti-proliferative effects when combined with hypomethylating agents and a BCL2 inhibitor. We previously discovered that CDC7 activity drives resistance to FLT3 inhibition (Cancer Cell. 2021 Jul 12;39(7):999-1014.e8), and now show that inhibition of CDC7 overcomes resistance to a variety of FLT3 inhibitor resistant cell lines. CDC7 inhibition is efficacious in multiple AML models resistant to standard of care agents, suggesting that CDC7 inhibitor combinations may be efficacious in the clinic. Results: We identified a potent and selective CDC7 inhibitor, SGR-2921, using large-scale physics-based computational modeling technology. In a panel of ~300 cancer cell lines, AML cell lines were on average the most sensitive to SGR-2921, and AML patient samples were highly sensitive to CDC7 inhibition ex vivo. In vivo, SGR-2921 showed strong anti-tumor growth activity in MV-4-11 and Molm-16 AML xenograft models at tolerated doses. In combination with hypomethylating agents, SGR-2921 increased the level of replication stress, DNA damage and apoptosis markers in vitro. Combination of SGR-2921 with venetoclax (BCL2 inhibitor) showed synergy on anti-tumor activity both in vitro and in vivo. SGR-2921 was highly efficacious in AML cell lines resistant to FLT3 inhibitors, hypomethylating agents and venetoclax, and in multi-agent resistant cell lines. Co-treatment with SGR-2921 partially restored sensitivity to FLT3 inhibition in FLT3 resistance AML cell lines. Conclusions: SGR-2921, a novel, potent CDC7 small molecule inhibitor, demonstrates strong anti-proliferative activity both in vitro in AML cell models and in vivo in AML xenograft models. SGR-2921 showed synergistic inhibitory effects on cell-proliferation and tumor growth in combination with standard of care agents, and was anti-proliferative in AML cell lines and patient samples resistant to standard of care agents. Together, these data show that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with a potential utility in patients with relapsed and refractory AML.

WEE1 inhibits the activation of both CDK1 and CDK2 through phosphorylation of Tyr15, allowing DNA damage repair before entering mitosis, thereby regulating the cell cycle in S and G2/M phases. Inhibition of WEE1 could result in premature progression through the G2/M cell cycle checkpoint with unresolved DNA damage, leading to mitotic catastrophe and cell death. Small molecule WEE1 inhibitors, such as AZD1775 and Zn-C3, are currently being evaluated in the clinic and have demonstrated promising efficacy in solid tumors including ovarian, colon, and uterine carcinoma. By applying Schrödinger’s computational platform including Free Energy Perturbation (FEP) and Protein FEP, we have identified novel, potent, and highly selective WEE1 inhibitors with IC50 values in the low nanomolar range in a biochemical kinase activity assay and cellular target engagement (CDK1 pTyr15) IC50s of 100 - 300 nM in A427 and OVCAR3 cell lines. The compounds also show potent anti-proliferative activity in over 20 breast and ovarian tumor cell lines, including cell lines insensitive to PARP inhibitors. The compounds demonstrate superior kinase selectivity compared to AZD1775 and Zn-C3 in a broad kinase panel with >450 kinases (ScanMAX). In addition, the compounds show desirable ADME properties and PK profiles in preclinical species. Based on in vitro CYP3A4 TDI assay performance (kinact/KI), we have reduced the potential for drug-drug interaction liabilities compared to AZD1775. In the A427 xenograft model, our WEE1 inhibitors demonstrate dose-dependent tumor growth inhibition and tumor regression at high doses. Anti-tumor activity is also demonstrated in additional tumor models, including OVCAR3 and HCC1806 xenograft models. The established PK-PD relationship shows sustained target engagement (pCDK1), increased DNA damage (gH2AX) and mitosis (pHH3). We demonstrate that hematological adverse effects can be mitigated by dosing holidays in xenograft tumor models while maintaining anti-tumor activity. Notably, our compound shows more sustained anti-tumor activity with dosing holidays compared to AZD1775, which we believe is attributable to the prolonged and higher exposure in tumor and plasma with our compound. In the A427 non small-cell lung cancer xenograft model, following 3 dosing holiday cycles at high doses, tumor eradication was maintained after treatment was stopped. In summary, we have identified novel, potent and exquisitely selective WEE1 kinase inhibitors that demonstrate robust anti-tumor activity and sustained target engagement in tumor models. The compound’s anti-tumor effects are maintained with dosing holidays while allowing full recovery of mechanism-based hematological effects. Citation Format: Shaoxian Sun, Sarah Silvergleid, Aleksey I. Gerasyuto, Jiashi Wang, Robert D. Pelletier, Andrew Placzek, Jennifer L. Knight, Anthony Clark, Hamish Wright, Wu Yin, Jackson Chief Elk, Jeff Bell, Pieter H. Bos, Nicholas A. Boyles, Eric Therrien, Kristian Jensen, Karen Akinsanya. Discovery of potent, selective, and orally available WEE1 inhibitors that demonstrate increased DNA damage and mitosis in tumor cells leading to tumor regression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2570.

Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a key mediator of the NF-κB signaling pathway, the main driver of a subset of B-cell lymphomas and functions by forming a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL). Previously, we described the discovery of novel and potent MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells. Here, we highlight the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models and the combination potential with agents including standard-of-care.

Abstract Introduction: CDC7 is a serine/threonine protein kinase that phosphorylates the MCM2-7 helicase complex, a required step in DNA replication initiation. CDC7 has emerged as an attractive target for cancer treatment because of high expression in a number of tumors (e.g. ovarian, lung, and oral) which is thought to be linked to their proliferative capacity and ability to bypass DNA damage responses. Consistent with this, disruption of CDC7 activity in cancer cells results in delayed DNA replication, mitotic abnormalities and cell death whereas non-transformed, p53 wildtype cells are protected from cytotoxicity due to G1 cell cycle arrest. Due to the low ATP Km of CDC7, very potent inhibitor molecules are required to effectively block CDC7 activity and drive cancer cells into apoptosis. We have identified novel potent and selective CDC7 inhibitors targeting the ATP binding site that are active in biophysical, biochemical and cellular assays as well as in vivo CDX models. Results: Our lead compounds show potent picomolar (pM) inhibition of CDC7 in a biochemical kinase activity assay, pM affinity in SPR assay and complete inhibition of MCM2 (S53) phosphorylation in COLO205, A427, MV-4-11 and SW48 cancer cell lines. In a broad kinase selectivity panel, the novel inhibitors showed good selectivity for CDC7 kinase. Mechanistic studies show that our CDC7 inhibitors induced apoptosis, disrupted DNA replication and cell cycle dynamics with accumulation of polyploid cells after 48 h of treatment of cancer cells with minimal effects on human fibroblast cell lines. Our compounds have shown potent anti-proliferative and cytotoxic effects in a panel of more than a 100 cancer cell lines of varying origin including COLO205, SW48, A427, MOLM-13, and SUM149. Comparison of CDC7 inhibitors with other oncology drugs in a panel of cancer cell lines revealed a unique mechanism of action. In vivo, our compounds reduced tumor cell MCM2 (S53) phosphorylation in the mouse COLO205 xenograft model and showed strong tumor growth inhibition. We have also examined the effect of CDC7 inhibitors on cancer cell proliferation in combination with other anti-cancer agents, including other DNA damage response (DDR) targeting agents. Conclusions: We have identified novel potent ATP-competitive CDC7 inhibitors that show target engagement in cells and CDX tumors and have shown strong inhibition of cancer cell proliferation in vitro and tumor growth in vivo. CDC7 inhibitors show promise for use in combination with other targeted therapies for the treatment of cancers of varying origin. Citation Format: Lyuben Tsvetkov, Adam Levinson, Xianhai Huang, Sayan Mondal, Jeff Bell, Lin Tang, Robert Pelletier, Karen Dingley, Nick Boyles, Jackson Chief Elk, Leah Frye, Alan Futran, Phani Ghanakota, Jeremy Greenwood, George Lai, Sarah Silvergleid, Wu Yin, Hamish Wright, Karen Akinsanya, Wayne Tang, Kristian Jensen. Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells [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 1277.

Introduction: Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that regulates several proteins involved in cell cycle, DNA repair, genomic stability, and epigenetics and has been implicated in cancer progression. A key substrate of USP7 is MDM2, the oncogenic E3 ubiquitin ligase that promotes degradation of the tumor suppressor p53. USP7-mediated stabilization of MDM2 leads to the degradation of p53, preventing cell cycle arrest and the induction of apoptosis and promoting tumor cell growth. In addition to the MDM2-p53 pathway, USP7 regulates a number of other substrates involved in cancer, including PIM2 kinase, MYCN, the DNA methyltransferase DNMT1, and the PTEN tumor suppressor. Consistent with its function promoting oncogenic signaling pathways, genetic and pharmacological inhibition of USP7 has been shown to inhibit growth of a number of tumor cell lines in vitro and in vivo, and this anti-tumor activity is significantly enriched among cells expressing wild type p53. Thus, inhibition of USP7 is a promising therapeutic strategy, especially in cancers carrying wild type p53 that can be reactivated by suppression of MDM2. Results: We have discovered a new class of potent and selective USP7 inhibitors. These compounds bind to USP7 and prevent deubiquitinase activity in biochemical activity assays with picomolar potency. Our compounds induce accumulation of p53 and exhibit IC50s below 50 nM in cell viability assays in Multiple Myeloma and Acute Myeloid Leukemia (AML) cell lines. In AML cell lines, our USP7 inhibitors strongly synergize with the approved Bcl-2 inhibitor venetoclax. Unlike MDM2 antagonists currently undergoing clinical trials in AML, these USP7 inhibitors do not lead to a significant increase in MDM2 levels, and our compounds result in a more modest induction of p53, both of which could provide significant safety benefits. We have demonstrated differential sensitivity to MDM2 antagonists and our USP7 inhibitors in select cell lines and show that this effect is dependent upon p53 induction. Our compounds are orally bioavailable with a desirable PK profile in mice and induce p53 in tumor cells in CDX mouse models of Multiple Myeloma. Conclusions: We have identified novel, potent, orally bioavailable USP7 inhibitors that lead to p53 accumulation and cytotoxicity in cancer cells. We demonstrate mechanistic differences between USP7 and MDM2-p53 antagonists, which may lead to a safety advantage. We observe strong synergy between USP7 inhibitors and an approved treatment. Our lead compounds have favorable drug-like properties, promising mouse PK profiles, and can induce p53 accumulation in mouse CDX tumor models. These data demonstrate the therapeutic potential of our USP7 inhibitors and reveal specific opportunities for their use in the treatment of Multiple Myeloma and AML. Citation Format: Alan Futran, Tao Lu, Katherine Amberg-Johnson, Xiaoxiao Yang, Saidi He, Jeffrey Bell, Sarah Boyce, Markus Dahlgren, Karen Dingley, Liping Fang, Heidi Koldsoe, Zef Konst, Fang-Yu Lin, Robert Pelleltier, Heng Qian, Mats Svensson, Michael Trzoss, Jie Xu, Shuping Xu, Zhaowu Xu, Engin Yapici, Yan Zhang, Li Xing, Takao Suzuki, Xianhai Huang, Jiayi Xu, Hamish Wright, Kristian Jensen, Wayne Tang, Tao Guo, Karen Akinsanya, David Madge. Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells [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 1338.

Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), was identified as a translocation protein fused with cIAP2 in mucosa-associated lymphoid tissue (MALT) B cell lymphomas. MALT1, a key mediator of NF-κB signaling and the main driver of a subset of B-cell lymphomas, functions via formation of a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 has been considered as a potential therapeutic target for several non-Hodgkin B cell lymphomas as well as chronic lymphocytic leukemia (CLL). Here, we describe the discovery of novel, potent MALT1 inhibitors that result in antiproliferative effects in non-Hodgkin B-cell lymphoma cells. Results: We have identified novel small molecule MALT1 inhibitors using our proprietary physics-based Free Energy Perturbation (FEP+) modeling technology. Our compounds show potent (sub nM) inhibition of MALT1 enzymatic activity, as well as high binding affinity (sub nM) to MALT1 protein measured by Surface Plasmon Resonance (SPR). BCL10 is a binding partner of MALT1 that is cleaved by MALT1 at the C-terminus. Our inhibitors were efficacious in a target engagement assay showing prevention of BCL10 cleavage in Activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines OCI-LY3 and OCI-LY10, which are Bruton tyrosine kinase (BTK) inhibitor ibrutinib-resistant and -responsive respectively. Our compounds are potent inhibitors of IL10 secretion in both OCI-LY3 and OCI-LY10 cells, which is consistent with the inhibition of NF-κB signaling. We also examined the effect of our MALT1 inhibitors on ABC-DLBCL cell proliferation. Our inhibitors demonstrated potent anti-proliferative effects in both OCI-LY3 and OCI-LY10 cell lines, as well as synergistic effects with ibrutinib in a BTKi sensitive ABC-DLBCL cell panel. Examinations of a protease panel and off-target safety screening panel, as well as in vivo high dose tolerability study showed our compound had excellent selectivity and significant safety margin. Plasma IL10 and tumor BCL10 have been identified as robust PD markers in PK/PD studies in both OCI-LY3 and OCI-LY10 tumor bearing mice. Dose-dependent tumor growth inhibition was observed after 3 weeks of treatment in OCI-LY3 xenograft model, with efficacy also observed in combination with venetoclax. Ongoing work: We are continuing to explore the synergistic effects of our compounds with BTK inhibitors in B-cell lymphoma mouse models. Preliminary data showed potent inhibition of IL-2 secretion in Jurkat cells from our compound treatment. Additional studies are ongoing to elucidate the role of MALT1 inhibition in Treg as well as Teffector cells in vitro and in vivo. Refinement of the current inhibitor series, using co-crystal structures, is in progress in preparation for further development of optimized molecules. Conclusion and Future Plans: We have identified novel potent MALT1 protease small molecule inhibitors that are efficacious in the in vitro B-cell lymphoma cell proliferation assays and in the in vivo B-cell lymphoma xenograft model. Our data suggest that targeting MALT1 may expand therapy options for patients with selected B-cell lymphomas, such as ABC-DLBCL. Our work provided insight into the anti-tumor efficacy of our inhibitors in B-cell lymphomas as single agent, and ongoing work will continue to assess the potential combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphoma. Disclosures Yin: Schrodinger: Current Employment, Current equity holder in publicly-traded company. Zhe:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Placzek:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Trzoss:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Krilov:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feng:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lawrenz:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Pelletier:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lai:Triplet Therapeutics: Current Employment, Current equity holder in private company. Bell:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Calkins:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Grimes:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Tang:Schrodinger: Current Employment, Current equity holder in publicly-traded company. McRobb:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Gerasyuto:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feher:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Mondal:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Jensen:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Wright:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Akinsanya:Schrodinger: Current Employment, Current equity holder in publicly-traded company.

FOSAMAX® PLUS D is a once‐weekly alendronate (ALN) 70‐mg / vitamin D3 (vit‐D3) 5600 IU combination tablet developed by Merck & Co., Inc. A single‐dose, open‐label, randomized, 2‐period, crossover study was conducted to test bioequivalence between FOSAMAX® PLUS D combination tablets and co‐administration of corresponding doses of ALN 70‐mg (FOSAMAX®) and vit‐D3 5600 IU (2x 2800 IU) as individual tablets in healthy male and female Asian subjects to support registration in Taiwan.Results: the 90%CI of the GMR for ALN AUC0‐∞ fell within the pre‐specified bioequivalence bounds of 0.8 to 1.25, but slightly exceeded 1.25 for ALN AUC0‐last and Cmax. The 90% CI of the GMR for vit‐D3, unadjusted for endogenous vit‐D3, was also contained within the pre‐specified bioequivalence bounds. Thus, the combination tablet was bioequivalent based on ALN AUC0‐∞ and vit‐D3 unadjusted for endogenous levels. However, it was not bioequivalent to co‐administration with respect to ALN AUC0‐last and Cmax .In conclusion, the small differences between the combination tablet and co‐administration of individual doses for ALN AUC0‐last and Cmax are not considered to be clinically significant, as slightly increased ALN concentrations are unlikely to meaningfully affect the safety profile. TFDA accepted these results and considered them supportive for registration of the combination tablet in Taiwan.