BaF3 Cells Research Articles

Rational design of 4-((6-phenoxypyrimidin-4-yl)amino)-N-(4-(piperazin-1-yl)phenyl)-1H-pyrazole-3-carboxamide (LT-540-717) as orally bioavailable FLT3 inhibitor

In recent years, fms-like tyrosine kinase 3 (FLT3) was confirmed as an exciting target for treatment of AML. However, resistance to FLT3 inhibitors caused by acquired point mutations in tyrosine kinase domain (TKD) have limited their sustained efficacious. Thus, there remains an unmet need to develop high-efficacy FLT3 inhibitors against both FLT3 internal tandem duplication (ITD) and FLT3 (TKD) mutations. Herein, we describe the discovery of compound LT-540-717 (32), a potent FLT3 inhibitor (IC50: 0.62 nM), starting from FN-1501. Compound 32 exhibited highly inhibitory activity against several acquired FLT3 mutations including FLT3 (ITD, D835V), FLT3 (ITD, F691L), FLT3 (D835Y) and FLT3 (D835V). Additionally, 32 displayed potent antiproliferative activity against FLT3-mutation driven BaF3 and AML cells. Oral administration of 32 (25 mg/kg, QD) significantly prohibited tumor growth (tumor-inhibition rate is 94.18%), and no obvious side effect was observed even when increasing dose to 50 mg/kg (tumor-inhibition rate is 93.98%). Furthermore, 32 showed an acceptable bioavailability (F = 33.3% in rat and 72.7% in beagles), a suitable half-life time (T1/2 = 3.5 h in rat and T1/2 = 11.1 h in beagles), and a satisfactory metabolic stability. In summary, these results show the therapeutic potential of 32 to become a new anti-AML drug, especially for AML harboring dual FLT3 (ITD, TKD) mutations.

Abstract LB123: Poorer outcomes in EGFR L858R-driven NSCLC treated with osimertinib may be addressed with novel combination of BLU-945 and osimertinib

Abstract Background: Osimertinib, a 3rd-generation (gen) EGFR tyrosine kinase inhibitor (TKI), is standard of care in front-line (1L) patients with advanced EGFR mutant NSCLC; however, not all subgroups may benefit equally. In the phase 3 FLAURA study, patients with exon 19 deletions (ex19del) had a median progression-free survival (mPFS) of 21.4 months (mo); patients with L858R had a shorter mPFS of 14.4 mo. Poorer outcomes with L858R have also been reported with other 3rd-gen TKIs aumolertinib and lazertinib. These patients are a potential poor-risk subgroup for these therapies. Here, we explored outcomes of patients with L858R-driven NSCLC using real-world datasets (RWDs), analyzed potential contributors to poorer outcomes, including co-mutation incidence and osimertinib potency for each mutation, and report preclinical proof of concept of combination treatment BLU-945, an investigational next-gen L858R inhibitor, with osimertinib. Methods: Two large RWDs were analyzed for survival outcomes in 1L osimertinib-treated patients with ex19del or L858R from MD Anderson Cancer Center (MDACC; n=105) and the clinical-genomic Guardant INFORM database (EGFR ctDNA baseline positive; n=1386). IC50s of osimertinib on EGFR mutations and wildtype were determined in BaF3 cells. Preclinical studies of combination BLU-945 and osimertinib were performed in L858R-driven BaF3 xenograft models. Results: Both RWDs confirmed poorer prognosis for 1L osimertinib-treated patients with L858R vs ex19del. MDACC cohort showed a 12-mo PFS rate of 63% for L858R (n=45) vs 82% for ex19del (n=60); mPFS was immature. Guardant INFORM cohort (ctDNA baseline positive) had a median time-to-treatment discontinuation of 8 mo for L858R (n=517) and 11.4 mo for ex19del (n=869), P=0.003. Poor prognosis factors (including TP53 mutations and co-mutation number) were not significantly different between L858R and ex19del; both had a similar number of off-target mutations in post-osimertinib samples. Strong association was found between osimertinib cellular IC50 and osimertinib clinical trial outcomes (mPFS). Osimertinib exhibited most clinical and cellular activity on ex19del, followed by L858R, then G719X, and then exon 20 insertions. BLU-945 in combination with osimertinib in a BaF3 L858R xenograft model demonstrated a longer duration of response vs osimertinib monotherapy. Conclusions: In both RWDs, 1L osimertinib-treated patients with L858R-driven NSCLC had poorer outcomes vs ex19del, consistent with osimertinib’s weaker activity on L858R. Preclinically, BLU-945 in combination with osimertinib increased L858R inhibition, resulting in more durable antitumor activity in L858R xenografts vs osimertinib alone, supporting rationale for combination treatment in patients with L858R mutations. This combination is being evaluated in 1L patients with L858R in the SYMPHONY study (NCT04862780). Citation Format: Yasir Y. Elamin, Tyler Rouskin-Faust, Nicole Zhang, Teresa Green, Aditya Dhande, Brenton G. Mar, John V. Heymach, Chiara Conti. Poorer outcomes in EGFR L858R-driven NSCLC treated with osimertinib may be addressed with novel combination of BLU-945 and osimertinib [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 LB123.

Abstract 4033: Pan-exon mutant KIT inhibitor DCC-3009 demonstrates tumor regressions in preclinical gastrointestinal stromal tumor models

Abstract Introduction: Gastrointestinal stromal tumors (GISTs) are typically driven by primary mutations in KIT exons 9 or 11. Heterogeneous drug-resistant secondary mutations arise in patients treated with FDA approved KIT inhibitors, including imatinib and sunitinib. Drug resistant secondary mutations are found at multiple regions in the ATP pocket (encoded by exons 13 and 14) or activation switch (encoded by exons 17 and 18) of KIT kinase. In addition, multiple drug-resistant clones can arise within a tumor or in metastatic tumor sites. An inhibitor that can broadly and potently inhibit the spectrum of KIT mutations is highly sought. Ripretinib has been FDA approved as a 4th line treatment for GIST and has broad activity against KIT mutations, including clinical potency in patients with mutations in KIT exons 11, 17, or 18. DCC-3009 was designed as a next generation KIT inhibitor that broadly and potently inhibits primary KIT mutations in exons 9 and 11 and secondary drug-resistant mutations across exons 13, 14, 17, and 18. DCC-3009 is a potent and selective inhibitor in enzyme and cell-based assays, and has demonstrated efficacy in xenograft models driven by drug resistant KIT mutations. Methods: DCC-3009 was tested for inhibition of KIT mutants using standard enzyme and cell-based assays. Levels of phosphorylated KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. KIT mutant xenograft or patient-derived xenograft models were performed at Crown Biosciences or Labcorp, AAALAC accredited facilities, with the approval of Animal Care and Use Committees. Results: In BaF3 cells transfected with KIT mutants, DCC-3009 was shown to potently inhibit the spectrum of known primary and secondary drug-resistant mutations in GIST. The pan-mutant KIT profile of DCC-3009 was shown in vitro to be superior to 2nd and 3rd line standard of care therapies sunitinib and regorafenib. DCC-3009 was selective for KIT when screened against a large panel of kinases. DCC-3009 has optimized pharmaceutical properties for oral administration. In pharmacokinetic/pharmacodynamic studies DCC-3009 achieved sufficient free drug levels to significantly inhibit drug-resistant KIT mutants for 12 hr post dose. In xenograft studies, treatment with DCC-3009 twice daily led to tumor regression in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Conclusions: DCC-3009 is a pan-exon mutant KIT inhibitor exhibiting high potency in KIT mutants in pre-clinical models spanning exons 9, 11, 13, 14, 17 and 18. In vivo, DCC-3009 exhibited efficacy in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Based on this profile, DCC-3009 has entered formal preclinical development. Citation Format: Bryan D. Smith, Subha Vogeti, Timothy M. Caldwell, Hanumaiah Telikepalli, Yu Mi Ahn, Gada Al-Ani, Stacie L. Bulfer, Andrew Greenwood, Cale L. Heiniger, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, William C. Patt, Max D. Petty, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Sihyung Yang, Yu Zhan, Bertrand Le Bourdonnec, Daniel L. Flynn. Pan-exon mutant KIT inhibitor DCC-3009 demonstrates tumor regressions in preclinical gastrointestinal stromal tumor models. [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 4033.

Abstract 4036: NMS-0963 is a novel potent, selective and orally available Syk inhibitor with promising preclinical activity in diffuse large B-cell lymphoma

Abstract The B-cell receptor (BCR) is a key survival molecule for normal B cells and for most B-cell malignancies, such as Chronic Lymphocytic Leukaemia (CLL) and Non-Hodgkin’s Lymphomas (NHL) including Diffuse Large B-cell Lymphomas (DLBCL). Small molecule inhibitors of key signaling kinases involved in the BCR pathway, such as the Btk inhibitor ibrutinib and the PI3Kdelta inhibitor idelalisib, have already demonstrated significant clinical activity. Spleen tyrosine kinase (Syk) is a non-receptor cytoplasmic tyrosine kinase that plays a fundamental role in BCR signaling initiation thus representing an additional potential therapeutic target for the inhibition of the BCR pathway. Here, we report the discovery and characterization of NMS-0963, a novel potent, selective and orally available Syk inhibitor, identified through an integrated medicinal chemistry and rational design approach. NMS-0963 showed potent inhibitory effect on Syk in an in vitro biochemical assay and displayed a good selectivity profile on a broad panel of kinases. On-target potency was confirmed in a cell-based assay using BaF3 cells engineered to express constitutively activated Syk, showing potent inhibition of Syk activity resulting in antiproliferative effect with IC50s in the low nanomolar range. Effective inhibition of BCR-mediated signaling pathway was observed in DLBCL-derived cell lines. NMS-0963 showed favourable ADME profile and good in vivo pharmacokinetic profiles after oral administration in mouse, rat and dog. Furthermore, NMS-963 demonstrated striking in vivo efficacy in tumor models of CD79/Myd88 mutated DLBCL, superior to competitors. Together with a permissive preclinical safety profile, these results support a rational for clinical development of NMS-0963 in DLBCL. Citation Format: Grazia Saturno, Michele Modugno, Paolo Orsini, Giovanni Cervi, Laura Buffa, Ilaria Motto, Nilla Avanzi, Marisa Montemartini, Fabio Gasparri, Gemma Texido, Arturo Galvani, Antonella Isacchi. NMS-0963 is a novel potent, selective and orally available Syk inhibitor with promising preclinical activity in diffuse large B-cell lymphoma. [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 4036.

Sitravatinib as a potent FLT3 inhibitor can overcome gilteritinib resistance in acute myeloid leukemia

BackgroundGilteritinib is the only drug approved as monotherapy for acute myeloid leukemia (AML) patients harboring FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation throughout the world. However, drug resistance inevitably develops in clinical. Sitravatinib is a multi-kinase inhibitor under evaluation in clinical trials of various solid tumors. In this study, we explored the antitumor activity of sitravatinib against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.MethodsGrowth inhibitory assays were performed in AML cell lines and BaF3 cells expressing various FLT3 mutants to evaluate the antitumor activity of sitravatinib in vitro. Immunoblotting was used to examine the activity of FLT3 and its downstream pathways. Molecular docking was performed to predict the binding sites of FLT3 to sitravatinib. The survival benefit of sitravatinib in vivo was assessed in MOLM13 xenograft mouse models and mouse models of transformed BaF3 cells harboring different FLT3 mutants. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of sitravatinib.ResultsSitravatinib inhibited cell proliferation, induced cell cycle arrest and apoptosis in FLT3-ITD AML cell lines. In vivo studies showed that sitravatinib exhibited a better therapeutic effect than gilteritinib in MOLM13 xenograft model and BaF3-FLT3-ITD model. Unlike gilteritinib, the predicted binding sites of sitravatinib to FLT3 did not include F691 residue. Sitravatinib displayed a potent inhibitory effect on FLT3-ITD-F691L mutation which conferred resistance to gilteritinib and all other FLT3 inhibitors available, both in vitro and in vivo. Compared with gilteritinib, sitravatinib retained effective activity against FLT3 mutation in the presence of cytokines through the more potent and steady inhibition of p-ERK and p-AKT. Furthermore, patient blasts harboring FLT3-ITD were more sensitive to sitravatinib than to gilteritinib in vitro and in the PDX model.ConclusionsOur study reveals the potential therapeutic role of sitravatinib in FLT3 mutant AML and provides an alternative inhibitor for the treatment of AML patients who are resistant to current FLT3 inhibitors.

ACC010, a novel BRD4 inhibitor, synergized with homoharringtonine in acute myeloid leukemia with FLT3-ITD.

Bromodomain-containing protein 4 (BRD4) inhibitors have been clinically developed to treat acute myeloid leukemia (AML), but their application is limited by the possibility of drug resistance, which is reportedly associated with the activation of the WNT/β-catenin pathway. Meanwhile, homoharringtonine (HHT), a classic antileukemia drug, possibly inhibits the WNT/β-catenin pathway. In this study, we attempted to combine a novel BRD4 inhibitor (ACC010) and HHT to explore their synergistic lethal effects in treating AML. Here, we found that co-treatment with ACC010 and HHT synergistically inhibited cell proliferation, induced apoptosis, and arrested the cell cycle in FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-positive AML cells in vitro, and significantly inhibiting AML progression in vivo. Mechanistically, ACC010 and HHT cooperatively downregulated MYC and inhibited FLT3 activation. Further, when HHT was added, ACC010-resistant cells demonstrated a good synergy. We also extended our study to the mouse BaF3 cell line with FLT3-inhibitor-resistant FLT3-ITD/tyrosine kinase domain mutations and AML cells without FLT3-ITD. Collectively, our results suggested that the combination treatment of ACC010 and HHT might be a promising strategy for AML patients, especially those carrying FLT3-ITD.

Discovery of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine derivatives as novel FLT3 covalent inhibitors for the intervention of acute myeloid leukemia.

Small molecule covalent drugs have proved to be desirable therapies especially on drug resistance related to point mutations. Secondary mutations of FLT3 have become the main mechanism of FLT3 inhibitors resistance which further causes the failure of treatment. Herein, a series of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine covalent derivatives were synthesized and optimized to overcome the common secondary resistance mutations of FLT3. Among these derivatives, compound F15 displayed potent inhibition activities against FLT3 (IC50 = 123 nM) and FLT3-internal tandem duplication (ITD) by 80% and 26.06%, respectively, at the concentration of 1 μM. Besides, F15 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines MOLM-13 (IC50 = 253 nM) and MV4-11 (IC50 = 91 nM), as well as BaF3 cells with variety of secondary mutations. Furthermore, cellular mechanism assays indicated that F15 inhibited phosphorylation of FLT3 and its downstream signaling factors. Notably, F15 could be considered for further development as potential drug candidate to treat AML.

Design, synthesis, and evaluation of pyrido.[3,4-b]pyrazin-2(1H)-one derivatives as potent FLT3 inhibitors

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as driver mutations in a subgroup of AML patients. Herein, we describe the design, synthesis, and biological evaluation of a novel series of potent pyrido.[3,4-b]pyrazin-2(1H)-one derivatives as FLT3 inhibitors. The compounds exhibited moderate to potent FLT3 kinase inhibitory potency and excellent antiproliferative activities against MV4-11 cells. Among them, compound 13 demonstrated the most potent kinase activity against FLT3-D835Y (IC50 = 29.54 ± 4.76 nM) and cellular potency against MV4-11 cells (IC50 = 15.77 ± 0.15 nM). Compound 13 also efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-D835V/F, FLT3-F691L, and FLT3-ITD/D835Y. Furthermore, compound 13 was metabolically stable in mouse liver microsomes. Moreover, the treatment with compound 13 led to robust inhibition of FLT3 autophosphorylation on Tyr589/591 in MV4-11 cells. In summary, our data demonstrated that 13 was worthy of further study for the treatment of AML.

Discovery of HCD3514 as a potent EGFR inhibitor against C797S mutation in vitro and in vivo.

Osimertinib (AZD9291), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), has significantly improved the survival of non-small cell lung cancer (NSCLC) patients with EGFRT790M mutation, the major mechanism of acquired resistance to first-generation EGFR TKI. However, resistance to AZD9291 arises eventually and EGFRC797S mutation was reported to be a major resistance mechanism. Thus, it is highly valuable to develop novel EGFR fourth-generation inhibitors targeting C797S mutation to override the acquired resistance. In this study, we identified HCD3514 as a novel EGFR fourth-generation inhibitors targeting C797S triple mutation. It strongly inhibited EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S mutations with IC50 values of 1.0 and 2.0 nM, respectively. HCD3514 dose-dependently inhibited the activation of EGFR in both engineered BaF3 cells and tumor cells harboring EGFRC797S triple mutant and thus effectively suppressed the proliferation of the cells. Moreover, HCD3514 caused a dose-dependent increase of apoptosis in C797S triple mutant cells accompanied by increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, HCD3514 induced tumor growth inhibition in EGFR19del/T790M/C797S xenograft model as a single oral agent by decreasing the activation of EGFR. In addition to EGFRC797S triple mutations, HCD3514 also potently and selectively inhibited EGFRT790M double mutations (L858R/T790M and 19del/T790M). Collectively, HCD3514 is a highly selective and potent EGFR inhibitor against EGFRC797S triple mutations as well as EGFRT790M double mutations and is confirmed potently anti-tumor activity in preclinical models.

Design and synthesis of selective FLT3 inhibitors via exploration of back pocket II.

Aim: The clinical benefits of FLT3 inhibitors against acute myeloid leukemia (AML) have been limited by selectivity and resistance mutations. Thus, to identify FLT3 inhibitors possessing high selectivity and potency is of necessity. Methods & results: The authors used computational methods to systematically compare pocket similarity with 269 kinases. Subsequently, based on these investigations and beginning with in-house compound 10, they synthesized a series of 6-methyl-isoxazol[3,4-b]pyridine-3-amino derivatives and identified that compound 45 (IC50: 103 nM) displayed gratifying potency in human AML cell lines with FLT3-internal tandem duplications mutation as well as FLT3-internal tandem duplications-tyrosine kinase domain-transformed BaF3 cells. Conclusion: The integrated biological activity results indicated that compound 45 deserves further development for therapeutic remedies for AML.

ASK120067 (limertinib) exerts pre-clinical anti-tumor activity by inhibiting EGFR exon20 insertion

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are classic strategies for the individualized treatment for patients with non-small cell lung cancer (NSCLC). However, EGFR exon20 insertion (EGFR 20ins) mutations, accounting for 6%–12% of all EGFR mutant cases in NSCLC, are generally resistant to the reversible EGFR TKIs (such as gefitinib and erlotinib), which makes them challenging drug-targets in lung cancer. In our previous study, we identified ASK120067 (limertinib) as a novel 3rd-generation EGFR TKI targeting EGFR T790M mutation with promising clinical activities. Here, we accessed the potency of ASK120067 on EGFR 20ins activation and evaluated its in vitro and in vivo anti-tumor activity against EGFR 20ins driven tumor models. We found that ASK120067 showed potent inhibitory activity on TKI-resistant EGFR 20ins kinase. In TKI-resistant EGFR 20ins-dependent BaF3 cells, it dose-dependently suppressed EGFR phosphorylation, impeded cell proliferation, and induced cell apoptosis with much superior efficacy to gefitinib and erlotinib. Moreover, oral administration of ASK120067 decreased the level of phospho-EGFR 20ins and caused significant tumor regression in EGFR 20ins BaF3 xenograft model. These results presented the pre-clinical anti-tumor efficacy of ASK120067 in EGFR 20ins models and highlighted the potential value of ASK120067 for the treatment of NSCLC patients harboring EGFR 20ins mutations.

In Vitro Evidence of Double Blockade of Asciminib with Reduced Dose of ATP-Binding Pocket Inhibitors in the Treatment of Chronic Myeloid Leukemia Harboring ABL1 Kinase Domain Mutation

Background Asciminib (ASC), a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, inhibiting BCR-ABL1 via allosteric binding. ASCEMBL trial showed superior efficacy of ASC to Bosutinib (BOS) with a higher molecular response rate, and a lower rate of discontinuation due to lack of efficacy (24.2% vs 35.5% at week 96). However, 24.2% of the patients still experienced lack of efficacy with ASC monotherapy. Also, out of the 39 patients who discontinued ASC due to lack of efficacy, 22 patients (56%) showed a new or persistent ABL1 kinase domain (KD) mutation either on ATP binding sites or Myristoyl pocket site. This finding suggests that ASC monotherapy is not always sufficient to overcome TKI-resistance particularly in those who already experienced multiple TKI failure. Accordingly, dual inhibition of ABL1 kinase protein with ASC combined with ATP binding pocket inhibiter (ABPI) is very attractive option to overcome TKI resistance. The remaining question is dose. A phase 1 study attempted to determine appropriate dose of ASC in combination with fixed dose of ABPIs including Imatinib (IMA), Dasatinib (DAS) and Nilotinib (NIL). But, this approach with fixed dose of ABPIs could add up toxicities not just from ABPIs but also from the combination of two drugs. Accordingly, a different strategy is to be developed to reduce the exposure to ABPI, while combining with ASC. In the present study, we simulated the combination treatment of ASC with ABPIs based on a clinically accessible fixed-dose for various ABL1 mutations to obtain answers to the following questions:Can combination of ASC with 4 different type of ABPIs be universally effective to suppress CML cell growth via dual blockade?Is there a specific ABPI for dual blockade in a specific ABL1 kinase domain mutation?What is the drug concentration of ABPIs for dual blockade when combined with fixed dose of ASC? Methods and Materials In order to simulate the combination treatment of ASC with ABPIs, the IC50 for ASC and ABPI measured from wild type K562 and BaF3BCR-ABL+cell lines were applied: ASC (3nM), IMA (150nM), BOS (10nM), DAS (0.8nM) and NIL (10nM) was applied in the wild type (WT) BaF3 cell lines and cell inhibition was assessed with WST-8 assay. 8 different BaF3 cell lines including wild type, G250E, Y253H, E255K, M244V, M351T, F317V and H369P mutant lines were treated with ABPI alone, ASC alone, and in combination by serial dilution based starting from the measured IC50 concentration, and the Dose-Response matrix was calculated and analyzed through SynergyFinder. Results A part of the results are summarized in the figure. As shown, with each ABPI at the baseline IC50 concentration in K562 cells, cell growth was inhibited to 59.8%, 56.9%, 61.5%, 59.9% and 71.6-79.4% with IMA, BOS, DAS, NIL and ASC (Fig A). When cell growth inhibition was assessed with combination treatment of reduced dose of ABPI and fixed dose of ASC (3nM), we observed that the combination treatment of ASC with ABPI can inhibit BaF3 cell growth even at a reduced concentration of ABPI from IC50 (Fig B). When combined of ABPI at baseline doses with 3nM ASC, 100% of cell inhibition was noted as expected. When treated with 3nM ASC with with 50% reduced dose of ABPI, 84.6-100% of cell inhibition was noted in WT, 99.2-99.6% in G250E, 96.8-99.4% in E255K mutant BaF3 cell lines, concluding that these cell lines were highly sensitive to double blockade; M351T and F317V BaF3 cell lines showed in the range between 30-77% of cell inhibition with a half dose of ABPI/baseline dose ASC, referring it intermediate sensitive cell lines; H369P, Y253F and M244V cell lines showed less effective cell inhibition compared to other cell lines, indicating the low sensitive cell lines. It implies that each cell line does have its own inherent profile of resistance to double blockade (Fig C). In addition, DAS and BOS seems to be better ABPIs for double blockade for intermediate to low sensitive mutations except the F317V mutation, which can be overcome with IMA or NIL combination. Conclusion The current study strongly suggests that 1) double blockade with reduced dose of ABPI in combination with fixed dose of ASC is effective to control CML cells with different ABL1 KD mutations. Each cell lines does have its inherent resistance level to double blockades, which needs to be further explored. A phase 1 study with reduced dose of ABPI in combination with fixed dose ASC is strongly warranted to define an optimal dose for combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Inhibitor of FLT3 and Its Drug-Resistant Mutants with Superior Activity to Gilteritinib in Molm-13 Preclinical Acute Myeloid Leukemia Xenograft Model

Acute Myeloid Leukemia (AML) is a complex heterogenous disease characterized by the aberrant growth of undifferentiated myeloid progenitor cells typically driven by a combination of epigenetic and signaling mutations. In particular, fms-like tyrosine kinase 3 (Flt3) internal tandem duplication (FLT3-ITD) is associated with poor relapse-free and overall survival. Several FLT3 inhibitors have been developed such as the first generation multikinase inhibitor Midostaurin which is approved for newly diagnosed FLT3 mutant AML in combination with induction chemotherapy. Second generation inhibitors such as gilteritinib are more selective and potent and are also approved for relapsed/refractory FLT3 mutant AML. Although initial response has been positive with this class of agents, prolonged remission is rarely achieved, and diverse mechanisms of resistance often arise with FLT3 inhibitors representing an ongoing challenge. Common mechanisms of resistance to FLT3 inhibitors includes the acquisition of tyrosine kinase domain (TKD) secondary mutations such as the gatekeeper F691L TKD mutation, or the emergence of additional mutations in alternative signaling pathways such as RAS/MAPK. Thus, it is important to continue developing novel inhibitors capable of circumventing these resistance mechanisms. Herein we describe 2082-0047, a novel tyrosine kinase inhibitor with sub-nanomolar potency against FLT3 mutant AML, including TKD mutations. Compound 2082-0047 is a highly selective pan-FLT3 kinase inhibitor, as in both scanMAX and KdELECT KINOMEscan™ Profiling assays (Eurofins DiscoverX) it has shown 400-1000 fold selectivity. In our initial screening, 2082-0047 was only effective in FLT3-ITD mutant cells, however the KINOMEscan™ Profiling data described above demonstrates that 2082-0047 is potent against FLT3, FLT3 ITD, D835H, D835V, D835Y, D835V, K663Q, N841I, and R834Q FLT3 TKD mutations. We confirmed this data in a cellular assay using BaF3 cell lines expressing the TKD mutations D835Y, D835H or F691L alone or with Flt3-ITD. Next, we confirmed using immunoblot analysis of phosphorylated Flt3 that 2082-0047 is a potent inhibitor of FLT3-ITD signaling (superior to gilteritinib) in FLT3-ITD mutant AML cell lines. Using wildtype B6 mice, we conducted pharmacokinetic (PK) studies and found that 2082-0047 had 79 % oral bioavailability, achieved 1.2 μΜ Cmax after 4 hours, and had half-life of 4.7 hours. Next, our absorption, distribution, metabolism and excretion (ADME) studies showed lack of CYP inhibition of major human isoforms. Overall, our PK studies demonstrated overall favorable drug like properties enabling in vivo investigation of therapeutic activity. Therefore, we proceeded with using the MOLM-13 disseminated xenograft model in NCG recipient mice to determine the overall survival of 2082-0047 compared to gilteritinib. We enrolled mice 10 days post engraftment to receive daily oral vehicle, 3, 10, 20 or 30 mg/kg of 2082-0047 or 30 mg/kg of gilteritinib and found that 10 and 20 mg/kg of 2082-0047 were well tolerated and superior to gilteritinib at 30 mg/kg (Figure 1). In summary, we describe a novel and potent inhibitor of FLT3, including both internal tandem duplication and drug-resistant tyrosine kinase domain (TKD) mutants, with promising preclinical activity which is superior to the most potent FDA approved FLT3 inhibitor gilteritinib in an aggressive AML in vivo model. Our non-GLP in vivo safety studies confirmed a broad therapeutic window of 2082-0047. Ongoing Investigational New Drug (IND) enabling studies will include comparing the effect of 2082-0047 to gilteritinib in an immunocompetent syngeneic AML murine model harboring both a Flt3-ITD and F691 TKD gatekeeper mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

1-Methyl-3-((4-(quinolin-4-yloxy)phenyl)amino)-1H-pyrazole-4-carboxamide derivatives as new rearranged during Transfection (RET) kinase inhibitors capable of suppressing resistant mutants in solvent-front regions

REarranged during Transfection (RET) is a validated target for anticancer drug discovery and two selective RET inhibitors were approved by US FDA in 2020. However, acquired resistance mediated by secondary mutations in the solvent-front region of the kinase (e.g. G810C/S/R) becomes a major challenge for selective RET inhibitor therapies. Herein, we report a structure-based design of 1-methyl-3-((4-(quinolin-4-yloxy)phenyl)amino)-1H-pyrazole-4-carboxamide derivatives as new RET kinase inhibitors which are capable of suppressing the RETG810 C/R resistant mutants. One of the representative compounds, 8q, potently suppressed wild-type RET kinase with an IC50 value of 13.7 nM. It also strongly inhibited the proliferation of BaF3 cells stably expressing various oncogenic fusions of RET kinase with solvent-front mutations, e.g. CCDC6-RETG810C, CCDC6-RETG810R, KIF5B-RETG810C and KIF5B-RETG810R, with IC50 values of 15.4, 53.2, 54.2 and 120.0 nM, respectively. Furthermore, 8q dose-dependently inhibited the activation of RET and downstream signals and obviously triggered apoptosis in Ba/F3-CCDC6-RETG810 C/R cells. The compound also exhibited significant anti-tumor efficacy with a tumor growth inhibition (TGI) value of 66.9% at 30 mg/kg/day via i. p. in a Ba/F3-CCDC6-RETG810C xenograft mouse model. Compound 8q may be utilized as a lead compound for drug discovery combating acquired resistance against selective RET inhibitor therapies.

Discovery of Hexahydrofuro[3,2-b]furans as New Kinase-Selective and Orally Bioavailable JAK3 Inhibitors for the Treatment of Leukemia Harboring a JAK3 Activating Mutant.

Janus kinase 3 (JAK3) is a potential target for the treatment of hematological malignancies. Herein, we report the discovery of a series of new orally bioavailable irreversible JAK3 kinase inhibitors. The representative compound 12n potently inhibited JAK3 kinase activity with an IC50 value of 1.2 nM and was more than 900-fold selective over JAK1, JAK2, and Tyk2. Cell-based assays revealed that 12n significantly suppressed phosphorylation of JAK3 and the downstream effectors STAT3/5 and also robustly restrained proliferation of BaF3 cells transfected with JAK3M511I activating mutation and human leukemia U937 cells harboring JAK3M511I with IC50 values of 22.9 and 20.2 nM, respectively. More importantly, 12n showed reasonable pharmacokinetic (PK) properties, and oral administration of 12n at a dose of 50 mg/kg twice daily led to tumor regression in a U937 cell inoculated xenograft mouse model. Thus, 12n represents a promising lead compound for further optimization to discover new therapeutic agents for hematological malignancies.

Abstract 3342: Discovery of potent and selective next-generation EGFR inhibitors with activity against single, double, and triple mutant EGFR variants including T790M and C797S

Abstract Introduction: EGFR activating mutations are observed in 10-50% of NSCLC patients and the common mutations (L858R [L] and exon 19 deletions [D]) are initially sensitive to first-, second-, and third-generation EGFR inhibitors (eg erlotinib [1G], afatinib [2G], and osimertinib [3G], respectively). However, on-target resistance is observed in a substantial percentage of patients, with T790M (T) and C797S (C) observed most frequently (post-1G/2G and post-3G, respectively). We set out to identify a next-generation inhibitor with 1) potent activity against all 8 major single (L and D), double (LT, DT, LC, DC), and triple (LTC, DTC) mutant variants, 2) selectivity over wild type (WT) EGFR, and 3) ability to penetrate the central nervous system (CNS). Experimental Procedures: Drug activity was determined by assessing effects in kinase assays and on tumor and BaF3 cell lines expressing WT or mutant forms of EGFR. For efficacy studies, mice were dosed orally once daily at doses that did not exceed the maximum tolerated dose. Results: Here we report the identification of a series of compounds with potent and selective in vitro and in vivo activity against all major classes of EGFR activating and resistance mutations and with selectivity over WT EGFR. A representative member from this series (Cmpd A) potently inhibits the kinase activity of EGFR L, LT, and LTC variants with IC50s ≤0.2 nM, compared to IC50s for osimertinib of 0.4, 0.2, and 360 nM respectively. In engineered BaF3 cells, Cmpd A inhibited viability of all 8 EGFR single, double, and triple mutant variants with IC50s <7 nM, while erlotinib was inactive against LT, DT, LTC, and DTC, and osimertinib was inactive against LC, DC, LTC, and DTC. Cmpd A inhibited the activity of all 8 variants with 7- to 46-fold selectivity over WT EGFR, which compared favorably to the 7- to 10-fold selectivity erlotinib exhibited for L and D. In vivo, Cmpd A (30 mg/kg QD) induced tumor regression, or inhibited tumor growth by at least 90%, in models containing EGFR single (L), double (LT), and triple (LTC and DTC) mutant variants. Studies to examine the CNS penetration of compounds are underway. Conclusions: We have identified a series of next-generation EGFR inhibitors with potent in vitro and in vivo activity against single, double, and triple mutant EGFR variants including T790M and C797S. Citation Format: Wei-Sheng Huang, Sara Nadworny, Narayana Narasimhan, Charles J. Eyermann, David C. Dalgarno, Victor M. Rivera, William C. Shakespeare. Discovery of potent and selective next-generation EGFR inhibitors with activity against single, double, and triple mutant EGFR variants including T790M and C797S [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 3342.

New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.

Identification of 2-Aminopyrimidine Derivatives as FLT3 Kinase Inhibitors with High Selectivity over c-KIT.

Herein, we report two promising compounds 30 and 36 possessing nanomolar FLT3 inhibitory activities (IC50 = 1.5-7.2 nM), high selectivity over c-KIT (>1000-fold), and excellent anti-AML activity (MV4-11 IC50 = 0.8-3.2 nM). Furthermore, these two compounds efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-ITD, FLT3-D835V/F, FLT3-F691L, FLT3-ITD-F691L, and FLT3-ITD-D835Y. Oral administration of 30 and 36 at 6 mg/kg/d could significantly suppress tumor growth in the MV4-11 cell-inoculated xenograft model, exhibiting tumor growth inhibitory rates of 83.5% and 95.1%, respectively. Importantly, 36 could prolong the mouse survival time in the FLT3-ITD-TKD dual mutation syngeneic mouse model (BaF3-FLT3-ITD-D835Y) at a dose of 6 mg/kg p.o. bid/4W. No clear myelosuppression was observed in the treated group of 36 in the MPO strain of zebrafish, even at 10 μM. In summary, our data demonstrated that 36 may represent a promising candidate for the treatment of FLT3 mutant AML.

The suppressive functions of Rora in B lineage cell proliferation and BCR/ABL1-induced B-ALL pathogenesis.

RORA plays an important role in regulating circadian rhythms, inflammation, metabolism and cellular development. Herein, we explore the roles of Rora in B cell proliferation and differentiation, as well as in Ph+ B-ALL. By using Roraloxp/loxp Mx-1-Cre mice, Rora was deleted in hematopoietic cells post Pipc induction. Rora deficiency mice were associated with an obvious accumulation of B cells in the peripheral blood, bone marrow, and spleen. On the other hand, activation of Rora with Cholesterol sulfate (CS) was associated with decreased B cell numbers. RNA-seq analysis revealed that the transcription level of Lmo1 was decreased in Rora deficient B cells. Moreover, the expression of RORA was shown to be decreased in Ph+ B-ALL cells compared to peripheral blood derived B cells from healthy donors. The overexpression of Rora in BaF3 cells with BCR/ABL1 was also associated with impeded the cell growth and an increased apoptotic rate compared to cells transduced with BCR/ABL1 alone. The co-expression of BCR/ABL1 and Rora induced B-ALL mouse model was associated with the significant inhibition of BCR/ABL1-transformed cell growth and prolonged the survival of the diseased mice. These results suggest a novel role for Rora in B cell development and Ph+ leukemogenesis.

LS-106, a novel EGFR inhibitor targeting C797S, exhibits antitumor activities both in vitro and in vivo.

With the wide clinical use of the third‐generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR‐mutated non–small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth‐generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS‐106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell‐free assay, LS‐106 potently inhibited the kinase activities of EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S with IC50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS‐106 exhibited comparable kinase inhibitory effect to osimertinib on EGFRL858R/T790M and wild‐type EGFR. Results from cellular experiments demonstrated that LS‐106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR19del/T790M/C797S or EGFRL858R/T790M/C797S, and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR19del/T790M/C797S (named PC‐9‐OR cells) using the CRISPR/Cas9 system and found that LS‐106 markedly suppressed the activation of EGFR19del/T790M/C797S and the proliferation of PC‐9‐OR cells. Moreover, cells harboring EGFR19del/T790M/C797S underwent remarkable apoptosis upon LS‐106 treatment. In vivo experiments further demonstrated that oral administration of LS‐106 caused significant tumor regression in a PC‐9‐OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS‐106 as a novel fourth‐generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S–triple‐mutant tumor models.