Supplementary Figure S1 shows mean (+SD) plasma concentrations of nemtabrutinib vs time following single (C1D1) oral doses of MK-1026 (linear and semi-log plots) nM

2068 Background: Epidermal growth factor receptor ( EGFR) gene is the most frequently altered oncogenic driver in glioblastoma (GBM). In-frame deletion alterations (e.g., EGFRvIII) and missense mutations co-occur in the setting of EGFR gene amplification and are characterized as a hallmark of disease pathogenesis in GBM. BDTX-1535 is an oral, highly potent, brain penetrant, selective, irreversible 4th generation tyrosine kinase inhibitor that targets EGFR alterations in GBM and NSCLC. Preliminary results of the Phase 1 dose escalation study (NCT05256290) of patients with recurrent GBM (rGBM) are presented here. Methods: BDTX-1535-101 is a first-in-human study that enrolled patients with either rGBM harboring EGFR alterations following standard of care or patients with locally advanced or metastatic EGFR mutated NSCLC that progressed on prior EGFR TKIs. Using an adaptive Bayesian optimal interval design in the Phase 1 part, patients were enrolled at increasing dose cohorts and were treated daily for 21-day cycles until treatment discontinuation. The primary objective was to determine the BDTX-1535 recommended Phase 2 dose based on the overall safety, PK, pharmacodynamics, and preliminary antitumor activity. Results: Twenty-seven patients with rGBM were enrolled in the Phase 1 cohort that consisted of 54 patients in total including 27 patients with NSCLC. Patients were treated across seven dose levels (15mg – 400mg QD). The mean age of patients with rGBM was 58.7 years (range 41-85) with 96% of patients with previous temozolomide (TMZ) treatment and a median of 2 lines of prior therapy (range 1-4). The most common all-grade treatment-related AEs across all cohorts were rash (78%), diarrhea (41%), fatigue (15%), stomatitis (11%), decreased appetite (11%), nausea (11%) and paronychia (11%). Gr 3 TRAEs ≥ 10% included rash (19%) reported at 300 or 400 mg QD doses. Plasma exposure of BDTX-1535 increased dose proportionally and had a half-life of ~15h, supporting once daily dosing. The maximum tolerated dose (MTD) is 300 mg QD. Among 19 evaluable patients for response based on RANO criteria, 1 confirmed partial response was observed and 8 patients achieved stable disease. Five patients remained on BDTX-1535 with stable disease for an extended period (>5 months) who previously performed poorly on TMZ with short treatment duration, and 1 patient continues on BDTX-1535 after 16 months of treatment. Conclusions: BDTX-1535 was well-tolerated up to 300mg daily, which is the MTD, and promising preliminary clinical activity was observed in patients with rGBM after relapse on standard of care treatment. Given the inability to reconfirm EGFR status at the time of treatment with BDTX-1535 in this Phase 1 trial, further exploration of BDTX-1535 in a “window of opportunity” study is ongoing (NCT06072586). Clinical trial information: NCT05256290 .

Abstract Nemtabrutinib is an orally bioavailable, reversible inhibitor of Bruton tyrosine kinase (BTK) and C481S mutant BTK. We evaluated the safety, pharmacology, and antitumor activity of nemtabrutinib in relapsed/refractory hematologic malignancies. Forty-eight patients with chronic lymphocytic leukemia (CLL), B-cell non–Hodgkin lymphoma (NHL), or Waldenström macroglobulinemia (WM), relapsed/refractory after ≥2 prior therapies were enrolled in the open-label, single-arm, phase I MK-1026-001 study (NCT03162536) to receive nemtabrutinib 5 to 75 mg once daily in 28-day cycles. Dose finding progressed using a 3 + 3 dose escalation design. Primary endpoints were safety and the recommended phase II dose (RP2D). Among 47 treated patients, 29 had CLL, 17 had NHL, and 1 had WM. Grade ≥3 treatment-emergent adverse events occurred in 37 (89%), most commonly neutropenia (11; 23.4%), febrile neutropenia (7; 14.9%), and pneumonia (7; 14.9%). The RP2D was 65 mg daily. An overall response rate of 75% was observed in patients with CLL at 65 mg daily. Significance: This first-in-human phase I study demonstrates the safety and preliminary efficacy of nemtabrutinib in patients with relapsed/refractory B-cell malignancies. These data support further exploration of nemtabrutinib in larger clinical studies. This article is featured in Selected Articles from This Issue, p. 5

Abstract Background: Epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in non-small cell lung cancer (NSCLC) and glioblastoma (rGBM). For NSCLC, EGFR tyrosine kinase activity can be driven by classical driver, intrinsic driver, and acquired resistance mutations. BDTX-1535 is an orally available, highly potent, brain penetrant, selective, irreversible small molecule tyrosine kinase inhibitor that targets all 3 classes of EGFR mutations. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Methods: BDTX-1535-101 (NCT05256290) is a Phase 1, open-label, multicenter study to assess the safety, tolerability, pharmacokinetics (PK), central nervous system (CNS) activity, and preliminary antitumor activity of BDTX-1535 in locally advanced or metastatic NSCLC with or without CNS disease or recurrent GBM (rGBM). The primary objective is to determine the BDTX-1535 recommended Phase 2 dose (RP2D) based on the overall safety, PK, pharmacodynamic, and preliminary antitumor activity. The monotherapy dose escalation portion evaluates BDTX-1535 in patients with locally advanced/metastatic NSCLC harboring intrinsic driver EGFR mutations following standard of care or with acquired resistance EGFR mutations following a 3rd generation EGFR inhibitor in the 1st line setting, in addition to patients with rGBM expressing EGFR alterations who previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). A capsule-to-tablet bridging cohort and a food effect cohort will also be assessed. Following the establishment of a preliminary recommended Phase 2 dose (RP2D), the disease-specific expansion portion of the study will further evaluate the safety, tolerability, and preliminary antitumor effect of BDTX-1535. Cohorts include the following:1) advanced/metastatic NSCLC with intrinsic resistance EGFR mutations following up to 2 lines of therapy, one of which must be an EGFR inhibitor treatment, 2) advanced/metastatic NSCLC with acquired resistance EGFR mutation following a 3rd generation EGFR inhibitor in the first line setting, 3) rGBM with confirmed EGFR alterations with or without amplifications. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with newly diagnosed GBM harboring EGFR alterations. Enrollment was initiated in 2022 and dose escalation is almost complete. Initiation of the dose expansion cohorts of NSCLC patients with EGFR acquired resistance and intrinsic driver mutations after progression on prior EGFR inhibitor is planned for the second half of 2023. For additional information, please contact ClinicalTrials@bdtx.com. Citation Format: Helena Yu, Melissa Johnson, Jason T Henry, Alex Spira, Ji-Youn Han, Minal Barve, James Battiste, Iyad Alnahhas, DoHyun Nam, Jeffrey Edenfield, Balazs Halmos, Shinkyo Yoon, Tae Min Kim, Sudharshan Eathiraj, Julio Hajdenberg, Sergey Yurasov, Manmeet Ahluwalia, Patrick Wen. A Phase 1 Study to assess BDTX-1535, an oral EGFR inhibitor, in patients with Glioblastoma or Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C036.

TPS9156 Background: The epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in many cancers, including glioblastoma (GBM) and non-small cell lung cancer (NSCLC). EGFR tyrosine kinase activity driven by common EGFR mutations can be inhibited by small molecules, however, resistance to available agents may be driven by mutations in the EGFR active kinase site or other regions. BDTX-1535 is an orally available, highly potent, selective, irreversible inhibitor of EGFR mutations, including extracellular variants and amplifications commonly expressed in GBM and inhibits the common and uncommon EGFR mutations found in NSCLC, including the C797S mutation acquired following 3 rd generation EGFR inhibitor therapy. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Preclinical studies suggest that BDTX-1535 has the potential to be clinically active in suppressing tumor growth in patients with GBM and NSCLC with or without CNS metastases, including a potential survival benefit. Methods: BDTX-1535-101 (NCT05256290) is Phase 1, open-label, multicenter study to assess the safety, tolerability, PK, CNS activity, and preliminary antitumor activity of BDTX-1535 in recurrent GBM (rGBM) or locally advanced or metastatic NSCLC with or without CNS disease. The Monotherapy Dose Escalation portion will evaluate BDTX-1535 in patients with either rGBM expressing EGFR alterations or locally advanced/metastatic NSCLC harboring sensitizing EGFR mutations with or without CNS disease. Patients with rGBM must have previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). Eligible NSCLC patients must have EGFR mutated NSCLC that has progressed following standard of care EGFR inhibitor therapy. Once a provisional recommended Phase 2 dose (RP2D) has been established, BDTX-1535 monotherapy will be explored in the following Dose Expansion cohorts to further evaluate safety, PK, and preliminary assessment of efficacy: 1) rGBM with confirmed EGFR alterations, 2) NSCLC with uncommon EGFR mutations following EGFR inhibitor therapy; 3) NSCLC with acquired EGFR resistance mutation following a 3rd generation EGFR inhibitor in 1L setting. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M or MET. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with rGBM harboring EGFR mutations or variants. Enrollment was initiated in 2022 and dose escalation is ongoing. Dose Expansion cohorts are expected to open in 2023. For additional information, please contact BDTX_1535_101_Study@bdtx.com . Clinical trial information: NCT05256290 .

Background: The epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in many cancers, including glioblastoma (GBM) and non-small cell lung cancer (NSCLC). EGFR tyrosine kinase activity driven by common EGFR mutations can be inhibited by small molecules, however, resistance to available agents may be driven by mutations in the EGFR active kinase site or other regions. BDTX-1535 is an orally available, highly potent, selective, irreversible inhibitor of EGFR mutations, including extracellular variants and amplifications commonly expressed in GBM and inhibits the uncommon EGFR mutations found in NSCLC, including the C797S mutation acquired following 3rd generation EGFR inhibitor therapy. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Preclinical studies suggest that BDTX-1535 has potential to be clinically active in suppressing tumor growth in patients with GBM and NSCLC with or without CNS metastases, including a potential survival benefit. Methods: BDTX-1535-101 (NCT05256290) is Phase 1, open-label, multicenter study to assess the safety, tolerability, PK, CNS penetrance, and preliminary antitumor activity of BDTX-1535 in recurrent GBM (rGBM) or locally advanced or metastatic NSCLC with or without CNS disease. The Monotherapy Dose Escalation portion will evaluate BDTX-1535 in patients with either rGBM expressing EGFR alterations or locally advanced/metastatic NSCLC harboring sensitizing EGFR mutations with or without CNS disease. Patients with rGBM must have previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). Eligible NSCLC patients must have EGFR mutated NSCLC that has progressed following standard of care EGFR inhibitor therapy. Once a provisional recommended Phase 2 dose (RP2D) has been established, BDTX-1535 monotherapy will be explored in the following Dose Expansion cohorts to further evaluate safety, PK, and preliminary assessment of efficacy: 1) rGBM with confirmed EGFR alterations, 2) NSCLC with uncommon EGFR mutations following EGFR inhibitor therapy; 3) NSCLC with acquired EGFR resistance mutation following a 3rd generation EGFR inhibitor in 1L setting. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M or MET. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with rGBM harboring EGFR mutations or variants. Enrollment was initiated in 2022 and dose escalation is ongoing. Dose Expansion cohorts are expected to open in 2023. For additional information, please contact BDTX_1535_101_Study@bdtx.com Citation Format: Melissa Johnson, Jason Henry, Alex Spira, James Battiste, Iyad Alnahhas, Manmeet Ahluwalia, Minal Barve, Jeffrey Edenfield, DoHyun Nam, Sudharshan Eathiraj, Julio Hajdenberg, Sergey Yurasov, Helena Yu, Patrick Wen. A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT127.

Acquired resistance to 3rd generation EGFR inhibitors used in the treatment of NSCLC is common. While the EGFR C797S substitution is a frequently reported post-osimertinib resistance mutation, real world evidence indicates the emergence of additional EGFR alterations that can drive drug resistance: kinase domain mutations (e.g., S768I), extracellular domain alterations (e.g., EGFRvIII, A289X), and EGFR amplification. Moreover, while osimertinib targets the classical EGFR mutations, primary NSCLC tumors are also driven by other oncogenic kinase domain mutations including G719X, S768I, and L861Q, which confer de novo (intrinsic) resistance to 3rd generation EGFR TKIs. A family of extracellular domain alterations occurs in nearly 50% of GBM patients, and these alterations are clinically resistant to all current generation inhibitors. Real world data in GBM demonstrate these EGFR alterations often co-occur and persist throughout treatment with current standard of care therapy. Of critical importance is the observation that the oncogenic isoform of EGFR in GBM is a covalent homo-dimer which can be formed and paradoxically activated by the binding of reversible 1st generation EGFR inhibitors. There is a significant clinical need to develop a potent CNS penetrant EGFR inhibitor that would target both acquired and intrinsic resistance mutations expressed in NSCLC and co-occurring EGFR alterations in GBM. An effective inhibitor should meet four design principles; 1) potent and selective against a broad family of intracellular, extracellular EGFR oncogenic alterations and amplification, 2) wild type EGFR sparing, 3) irreversible to avoid paradoxical activation, and 4) CNS penetrant. BDTX-1535 is a 4th generation irreversible CNS penetrant EGFR MasterKey inhibitor targeting a family of oncogenic EGFR extracellular domain alterations and amplification in GBM patients and EGFR resistance mutations in NSCLC. BDTX-1535 meets all four criteria for a highly effective EGFR inhibitor and was designed using the MAP Discovery Engine to target the activated conformations used by oncogenic EGFR to drive tumorigenesis. BDTX-1535 achieves potent anti-tumor activity against EGFR alterations and amplification across models including NSCLC and GBM PDX and intracranial tumors. BDTX-1535 is currently in a phase I clinical study in patients with NSCLC and GBM harboring sensitive EGFR alterations (NCT05256290). Citation Format: Matthew O'Connor, Matthew Lucas, Sherri Smith, Anthony Trombino, Sudharshan Eathiraj, Elizabeth Buck. Discovery of BDTX-1535, a novel 4th generation, irreversible, potent, wild type sparing EGFR MasterKey inhibitor that targets oncogenic kinase domain mutations as well as extracellular domain alterations for the treatment of NSCLC and GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3396.

Abstract Epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in non-small cell lung cancer (NSCLC) and glioblastoma (r/rGBM). For NSCLC, EGFR tyrosine kinase activity can be driven by classical driver (L858R, Ex19del), intrinsic (atypical) driver (L718Q, L861Q, S768I), and acquired resistance mutation (C797X). BDTX-1535 is an oral, highly potent, brain penetrant, selective, irreversible 4th generation tyrosine kinase inhibitor that targets all 3 classes of EGFR mutations. Preliminary results from the Phase 1 dose escalation study (NCT05256290) are presented. Methods: BDTX-1535-101 is a first-in-human study enrolling patients with locally advanced or metastatic NSCLC harboring EGFR mutations, with or without CNS disease, following standard of care EGFR inhibitor therapy, or patients with r/rGBM expressing EGFR alterations. The primary objective is to determine the BDTX-1535 recommended Phase 2 dose (RP2D) based on the overall safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity. Using an adaptive Bayesian optimal interval design, eligible patients are enrolled and dosed daily for 21-day cycles until treatment discontinuation. Results: As of 20 May 2023, 51 patients (24 NSCLC, 27 r/rGBM) were enrolled and treated across 7 dose cohorts (15mg – 400mg QD). For NSCLC, mean age was 65 years (range 47-81), patients had a median of 2 prior lines of treatment (1-9), and 79% received prior osimertinib. For r/rGBM, the mean age was 58 years (41-85) with a median of 2 prior lines of treatment (range 1-4). The most common all-grade treatment-related AEs (TRAE) were rash (76%), diarrhea (51%), stomatitis (29%), paronychia (20%) and nausea (20%), vast majority Gr 1 to Gr 2. Gr 3 TRAEs greater than or equal 10% were diarrhea (12%) and rash (10%). Plasma exposure of BDTX-1535 increased dose proportionally with a half-life of ~15h supporting once daily dosing. The maximum tolerated dose is 300mg QD. Circulating tumor DNA (ctDNA) assessment in NSCLC patients with classical and intrinsic drivers as well as acquired resistance mutations revealed significant ctDNA reductions across all EGFR mutation types. Five of 12 patients with either intrinsic driver (eg, L718Q) or acquired resistance (C797S) mutations in a subgroup of NSCLC efficacy evaluable patients at doses at or above 100mg had a confirmed partial response (PR) by RECIST1.1; 1 patient had unconfirmed PR, while the remaining 6 patients had stable disease. Additional GBM data will be shared separately.Conclusions:BDTX-1535 was well-tolerated at a daily dose up to 300mg and promising preliminary clinical activity was observed in NSCLC patients harboring either intrinsic driver or acquired resistance EGFR mutations post-progression with an EGFR inhibitor. A preliminary RP2D dose is being explored in NSCLC patients in dose expansion cohorts. : Citation Format: Helena Yu, Melissa Johnson, Jason T. Henry, Alex Spira, Ji-Youn Han, Minal Barve, James Battiste, Iyad Alnahhas, DoHyun Nam, Jeffrey Edenfield, Balazs Halmos, Shinkyo Yoon, Tae Min Kim, Sudharshan Eathiraj, Julio Hajdenberg, Sergey Yurasov, Manmeet Ahluwalia, Patrick Wen. Phase 1 study of BDTX-1535, an oral 4th generation inhibitor, in patients with Non-Small Cell Lung Cancer and Glioblastoma: Preliminary dose escalation results [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C022.

Inhibitors of B cell receptor (BCR) signaling such as the Bruton's tyrosine kinase (BTK) inhibitors are effective therapeutics for chronic lymphocytic leukemia (CLL). The first-in-class covalent BTK inhibitor, ibrutinib, produces durable responses in most CLL patients; however, complete responses are only observed in a minority of patients. B cell lymphoma 2 (BCL2), an anti-apoptotic protein that contributes to CLL cell survival, has also been investigated as a therapeutic target. The BCL2 inhibitor venetoclax is effective in patients with CLL and can produce undetectable minimal residual disease, allowing discontinuation of therapy. In combination, ibrutinib and venetoclax have shown preclinical synergy and clinical efficacy. Nemtabrutinib is a next generation, reversible inhibitor of BTK that potently inhibits BCR signaling in treatment-naïve and ibrutinib-refractory CLL cells ex vivo. The clinical efficacy of combining BTK inhibitors with BCL2 inhibitors motivated us to evaluate the novel combination of nemtabrutinib and venetoclax. In vitro studies show that nemtabrutinib and venetoclax are not antagonistic to each other. In an adoptive transfer CLL mouse model, mice treated with nemtabrutinib and venetoclax had prolonged survival compared to mice treated with ibrutinib and venetoclax. Our preclinical studies further validate the combination of BTK inhibitors with venetoclax and justify further investigation of combining nemtabrutinib with venetoclax in CLL.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton’s tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

Abstract NSCLC accounts for approximately 85% of lung cancer cases worldwide. NSCLC harboring EGFR mutations constitutes 10-20% of all lung cancer cases in Europe and North America, and up to 50% of those in Asia. The majority (80-90%) of these mutations are either Exon19del or L858R. Uncommon EGFR mutations, of which G719X, S768I and L861Q are amongst the most frequent, account for 10-20% of EGFR mutations in NSCLC. Additionally, secondary EGFR mutations such as C797S that emerge during treatment with osimertinib occur in ~10% of patients. Current generation EGFR inhibitors with efficacy against common, uncommon and/or resistance mutations are either poorly brain penetrant or do not have broad spectrum activity against multiple mutations. BDTX-1535 is an irreversible, spectrum selective MasterKey inhibitor of common, uncommon and resistance EGFR mutations such as G719X and C797S that occur in NSCLC (IC50<10nM). BDTX-1535 is differentiated from many EGFR inhibitors by its CNS-penetrating properties. BDTX-1535 has a Kpuu, defined as the ratio of the unbound brain tissue concentration over the unbound plasma concentration, of 0.8 in rat. In studies of EGFR Exon19del+C797S, BDTX-1535 achieved pEGFR suppression exceeding 24 hours in vitro and following a single dose in vivo. BDTX-1535 has demonstrated robust tumor growth inhibition and regressions in multiple pre-clinical models, including PDX intracranial models. Thus, BDTX-1535 has potential to treat patients with NSCLC harboring a broad range of mutations, both common and uncommon, as well as those associated with resistance to the current standard of care TKIs. The CNS penetrating properties may help to treat CNS metastases or to prevent them from occurring. BDTX-1535 is currently being evaluated in IND-enabling studies. Citation Format: Matthew C. Lucas, Melinda S. Merchant, Matthew O'Connor, Carl Cook, Sherri Smith, Anthony Trombino, Wu-Yan Zhang, Irache Visiers, Kate Tith, Reza Foroughi, Nigel Waters, Iwona Wrona, Michael Pickard, Sudharshan Eathiraj, Karsten Witt, Christopher Roberts, Rachel Humphrey, Elizabeth Buck. BDTX-1535, a CNS penetrant MasterKey inhibitor of common, uncommon and resistant EGFR mutations, demonstrates in vivo efficacy and has potential to treat osimertinib-resistant NSCLC with or without brain metastases [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P02-04.

Background Acute myeloid leukemia (AML) is the most common type of adult leukemia. Several studies have demonstrated that oncogenesis in AML is enhanced by kinase signaling pathways such as Src family kinases (SFK) including Src and Lyn, spleen tyrosine kinase (SYK), and bruton's tyrosine kinase (BTK). Recently, the multi-kinase inhibitor ArQule 531 (ARQ 531) has demonstrated potent inhibition of SFK and BTK that translated to improved pre-clinical in vivo activity as compared with the irreversible BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) models. Given the superior activity of ARQ 531 in CLL, and recognition that this molecule has a broad kinase inhibition profile, we pursued its application in pre-clinical models of AML. Methods The potency of ARQ 531 was examined in vitro using FLT3 wild type and mutated (ITD) AML cell lines and primary samples. The modulation of pro-survival kinases following ARQ 531 treatment was determined using AML cell lines. The effect of SYK expression on ARQ 531 potency was evaluated using a SYK overexpressing cell line (Ba/F3 murine cells) constitutively expressing FLT3-ITD. Finally, the in vivo activity of ARQ 531 was evaluated using MOLM-13 disseminated xenograft model. Results Our data demonstrate that ARQ 531 treatment has anti-proliferative activity in vitro and impairs colony formation in AML cell lines and primary AML cells independent of the presence of a FLT3 ITD mutation. We demonstrate decreased phosphorylation of oncogenic kinases targeted by ARQ 531, including SFK (Tyr416), BTK, and fms-related tyrosine kinase 3 (FLT3), ultimately leading to changes in down-stream targets including SYK, STAT5a, and ERK1/2. Based upon in vitro drug synergy data, we examined ARQ 531 in the MOLM-13 AML xenograft model alone and in combination with venetoclax. Despite ARQ 531 having a less favorable pharmacokinetics profile in rodents, we demonstrate modest single agent in vivo activity and synergy with venetoclax. Conclusions Our data support consideration of the application of ARQ 531 in combination trials for AML targeting higher drug concentrations in vivo.

Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.

Introduction: Resistance to covalent BTK inhibitors such as ibrutinib and acalabrutinib is a common mechanism of resistance that portends a poor long-term clinical outcome. ARQ 531 is a potent, reversible inhibitor of both wild type and ibrutinib-resistant C481S-mutant BTK. ARQ 531 suppresses oncogenic BCR signaling in CLL cells resistant to ibrutinib and has demonstrated antitumor activity superior to ibrutinib in CLL, Richter's transformation, and DLBCL mouse models. Methods: The primary objectives of the clinical study were to assess the safety and tolerability of ARQ 531, and to determine the recommended Phase 2 dose (RP2D) and schedule. The secondary objectives were to assess the pharmacokinetic (PK) profile, pharmacodynamic (PD) activity, and preliminary evidence of anti-tumor activity. Eligible patients had relapsed/refractory CLL/SLL, B-cell NHL or Waldenstrom's macroglobulinemia, had received at least 2 prior lines of systemic therapy and had good organ function including creatinine clearance ≥ 60 mL/min as estimated by the Cockcroft-Gault equation or by 24-hour urine collection, absolute neutrophil count ≥ 1000/µL, platelet count ≥ 50,000/µL and hemoglobin ≥ 8.0 g/dL. Prior therapy for CLL must have included an irreversible BTK inhibitor. Dose escalation was performed according to a 3+3 study design. Treatment emergent adverse events (TEAEs) were assessed per NCI CTCAE v.4.03. Tumor responses were evaluated per disease specific guidelines. Results: As of July 19, 2019, a total of 40 patients have been treated: CLL/SLL (n=26), Richter's transformation (n=6), DLBCL (n=3), FL (n=4), MCL (n=1). Baseline demographics were: median age 65.5 (range 47-82) years, male/female 36/4 and median number of prior lines of therapy 4 (range 2-12). BTK-C481S mutation was documented in 22/26 (85%) CLL patients. Enrolled patients received ARQ 531 orally once daily, continuously, in 28- day cycles at doses of 5, 10, 15, 20, 30, 45, 65 and 75 mg QD. The most common drug-related TEAEs that occurred in > 2 patients were nausea (n=4), diarrhea (n=4), fatigue (n=3), neutrophil count decreased (n=3), dysgeusia (n=3) and rash (n=3). The majority of the drug-related TEAEs were grade 1 or 2. Drug-related grade 3 or 4 TEAEs included neutrophil count decreased (n=3), as well as febrile neutropenia, cellulitis, platelet count decreased, lipase increased, and rash (one each). One subject treated at 65 mg experienced a DLT of grade 3 rash. The 65 mg cohort was expanded to a total of 10 patients, and no other DLTs were observed. At the 75 mg QD dose level (n =4), drug-related grade 2 adverse events were reported which led to dose reduction to 65 mg QD (n=3) or treatment discontinuation (n=1). Preliminary PK data show that patients receiving ARQ 531 at 65 mg QD exhibit steady-state trough concentrations (Cmin) above 1 µM; the estimated plasma half-life generally ranged from 20-30 hours and was associated with complete pBTK inhibition. Clinical responses to ARQ 531 were observed in multiple patients with B-cell malignancies. Ten partial responses (PRs) were achieved mainly in the higher dose cohorts, and included patients with CLL (n=7), Richter's transformation (n=1), DLBCL (n=1) and follicular lymphoma (n=1). Of the 7 CLL patients who attained PRs, 5 were initiated at 65 mg, 1 was initiated at 45 mg and was dose escalated to 65 mg and 1 was initiated at 75 mg and dose reduced to 65 mg. Together, the safety, PK/PD and clinical activity results suggest that ARQ 531 at 65 mg QD is safe, well tolerated and has clear signs of anti-tumor efficacy. Thus, 65 mg QD dose has been selected as the RP2D in patients with B-cell malignancies. Conclusion: ARQ 531 has a manageable safety profile and shows anti-tumor activity as single agent therapy in heavily pre-treated patients with B-cell NHL and in patients with CLL resistant to covalent BTK inhibitor. The Phase 1 dose escalation portion of this study is complete. Enrollment of patients with multiple B-cell malignancies is ongoing at 65 mg QD in the phase 1b expansion portion of the study. Updated safety, PK, biomarker and anti-tumor activity data will be presented. Disclosures Woyach: Morphosys: Research Funding; Verastem: Research Funding; Loxo: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding. Stephens:Karyopharm: Research Funding; Gilead: Research Funding; Acerta: Research Funding. Flinn:F. Hoffmann-La Roche Ltd: Research Funding; TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; AbbVie, Seattle Genetics, TG Therapeutics, Verastem: Consultancy; TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; Acerta Pharma, Agios, Calithera Biosciences, Celgene, Constellation Pharmaceuticals, Genentech, Gilead Sciences, Incyte, Infinity Pharmaceuticals, Janssen, Karyopharm Therapeutics, Kite Pharma, Novartis, Pharmacyclics, Portola Pharmaceuticals: Research Funding. Bhat:Pharmacyclics: Consultancy; Janssen: Consultancy. Savage:ArQule, Inc.: Employment. Chai:ArQule, Inc.: Employment. Eathiraj:ArQule, Inc.: Employment. Granlund:ArQule, Inc.: Employment. Schwartz:ArQule, Inc.: Employment. Byrd:Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding.

words: 198 Text words: 3887 Number of Figures: 7 Number of Tabs: 1 References: 61

Dysregulation of the PI3K-AKT signaling pathway has been implicated as a key driver in cancer initiation and progression. AKT is a serine/threonine kinase and a critical component mediating the PI3K-AKT signaling axis. Although AKT inhibitors have been extensively studied, clinical outcome has not been impressive. Interestingly, it has been shown that PI3K/AKT pathway has been involved in resistance to conventional chemotherapy, and inhibition of AKT enhances targeted therapy and sensitizes radiation therapy. Miransertib is a potent and selective pan-AKT inhibitor and currently in early clinical studies. In this study, we assessed combined effect of Miransertib with immune checkpoint inhibitor, anti-MEK and anti-HER2 agents, and a chemotherapeutic agent in vivo. Miransertib at doses of range from 20mg/kg to 120mg/kg was tested in combination with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab, or paclitaxel. Anti-tumor efficacy was assessed in syngeneic mouse CT-26 colon and 4T1 breast tumor models and xenografts models with endometrial (AN3CA) and breast (HCC1954, KPL-4, ZR-75-1) tumors, two patient-derived (PDX) models of endometrial cancer (with PIK3CAH1047R and R93W+D350G mutations) and in one vemurafenib-resistant melanoma PDX (with BRAFV600E and PIK3CA H1047R mutations). Combinability and efficacy of Miransertib and anti-PD-1 antibody was assessed in syngeneic mouse CT-26 colon tumor model. Miransertib at 60 mg/kg and anti-PD-1 antibody at 10mg/kg were combinable. Combined dosing of Miransertib with Anti-PD-1 antibody exerted superior anti-tumor activity in comparison to the single agents (TGI: 65% for combination, 50% for Miransertib and 55% for anti-PD-1 antibody after dosing for 9 days and more significant difference after 12 day dosing). In 4T1 breast tumor model, combination of Miransertib and anti-PD-1 antibody showed a very modest anti-tumor activity whereas there is no effect as single agents. In one of the endometrial PDX models, the combination of Miransertib with trametinib enhanced anti-tumor activity of each drug alone, reducing tumor growth by 67% compared to single-agent tumor reductions by 43% for either Miransertib or trametinib. In a vemurafenib-resistant melanoma PDX model, tumor growth was reduced by 73% when Miransertib was combined with trametinib, while trametinib alone only reduced tumor growth by 26% for trametinib and by 16% by Miransertib. Miransertib enhanced efficacy of in combination with trastuzumab, lapatinib, or paclitaxel, by reducing tumor growth as much as 92%, 73% and 85% respectively, while single agent comparators reduced tumors by less than 50%. Miransertib is combinable with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab, and paclitaxel and exhibits enhanced anti-tumor activity. These results provide us rationale for the combination study of Miransertib in a clinical setting. Citation Format: Yi Yu, Terence Hall, Sudharshan Eathiraj, Ron E. Savage, Brian Schwartz. In vivo combination of miransertib (ARQ 092) with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab and paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2943.

Acute Myeloid Leukemia (AML) is a rapidly progressing hematopoietic malignancy arising from bone marrow myeloid progenitor cells. Treatment with cytotoxic chemotherapy has not changed for over four decades resulting in poor survival. The dismal prognosis could be attributed to the heterogeneity of this disease, where multiple genetically aberrant clones exist within the same patient. Mutations in the FMS-like tyrosine kinase (FLT3) occurs in 30% of AML patients, typically as an internal tandem duplication (ITD) resulting in a constitutively active FLT3 survival pathway. This has prompted the generation of selective FLT3 inhibitors such as Quizartinib and Gilteritinib which are currently being pursued in clinical trials. Still, acquired resistance to these selective FLT3 inhibitors due to the acquisition of tyrosine kinase domain mutations (TKD) can occur. This suggests that the use of an agent with a broader kinome inhibition profile (such as the recently granted FDA approved Midostaurin) could achieve more durable clinical benefit. ARQ 531 is a novel potent BTK inhibitor currently being investigated in a Phase 1 trial in patients with relapsed/refractory hematological malignancies (ClinicalTrials.gov Identifier: NCT03162536). We have found that ARQ 531 also has inhibitory activity against members of the Src family of kinases (SFK; including downstream target SYK) as well as FLT3. SYK directly binds to and trans-activates FLT3 which is essential for FLT3-ITD tumorigenicity, suggesting that ARQ 531 has therapeutic potential in AML. Therefore we have investigated the in vitro and in vivo efficacy of ARQ 531 in AML. Our preliminary studies demonstrate cytotoxicity for ARQ 531 in patient-derived primary AML cells harboring FLT3 wild type and FLT3-ITD, as well as multiple AML cell lines. Importantly, ARQ 531 is effective in a MOLM-13 tyrosine kinase inhibitor (TKI) resistant cell line harboring a FLT3-ITD-TKD-D835Y mutation. Furthermore, we show that ARQ 531 can reduce the level of phosphorylated FLT3, but unlike selective FLT3 inhibitors, it can also inhibit Src family phosphorylation and SYK phosphorylation. Additionally, ARQ 531 exhibited an anti-clonogenic effect on primary patient blasts using Methocult colony forming unit assay. Finally, to investigate the in vivo effect of ARQ 531, we used an aggressive AML MOLM-13 disseminated xenograft mouse model. NSG mice were randomized one-week post engraftment to either vehicle or daily oral gavage of 50 mg/kg ARQ 531. The estimated median survival for the ARQ 531 group was 23 days compared to 21 days for the vehicle group (p = 0.002) suggesting in vivo efficacy for ARQ 531 in AML. Collectively, we provide for the first time promising preclinical efficacy for ARQ 531 in AML supporting further mechanistic investigation of this agent, and potentially, expansion of the ongoing clinical studies to include AML patients. E. H. and J.C. B. contributed equally as co-senior authors to this work Citation Format: Ola A. Elgamal, Bridget Carmichael, Amy Lehman, Shelley J. Orwick, Minh Tran, Virginia M. Goettl, Shaneice Mitchell, Rosa Lapalombella, Jae Yoon Jeon, Sharyn D. Baker, Sudharshan Eathiraj, Brian Schwartz, Erin Hertlein, John C. Byrd. Preclinical evaluation of the tyrosine kinase inhibitor ARQ 531 in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1882.