Juxtamembrane Domain Research Articles

A study of the synthetic route and resistance of gilteritinib

Gilteritinib is used to treat acute myeloid leukemia (AML) carrying FMS-like tyrosine kinase 3 (FLT3) mutations. As it is a new drug, there are still many aspects that need improvement. This article will be written according to the following three points: Analysis of its mechanism of action with the receptor; Analysis of the three main synthetic routes of gilteritinib and reasonable suggestions are given; Causes and solutions to the emergence of drug resistance. The following 4 points have been found that:(1) FLT3-internal tandem duplication (ITD) mutation and FLT3-tyrosine kinase domain (TKD) mutation, leukemic cells' ability to proliferate, differentiate, and survive aberrant changes due to both kinds of mutations. (2) Gilteritinib combines with FLT3's ATP pocket, and the compound shaped from FLT3 to gilteritinib was synthesized by the juxtamembrane (JM) domain. (3) Synthesis route 3 in this paper is most suitable for industrial mass production. (4) Resistance to gilteritinib is divided into primary resistance and secondary resistance, research in the future should focus on finding new therapeutic targets.

T-Cell Receptor-Engineered T Cells Targeting FLT3-D835 Mutation-Derived Neoantigens in Acute Myeloid Leukemia

The adoptive transfer of T-cell receptor (TCR)-engineered T cells has demonstrated a remarkable anti-leukemic potential in mice models and human research for acute myeloid leukemia (AML). Neoantigens arising from somatic mutations are ideal targets for immunotherapy due to their high immunogenicity and harmless to normal cells. Therefore, neoantigen-specific TCR-based immunotherapy may have great prospects for AML. Activating mutations in FMS-like tyrosine kinase 3 ( FLT3) represent the most frequent genomic alterations of AML, including internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain, mostly at the activation loop residue D835. A number of FLT3 inhibitors have been clinically employed for FLT3-mutated AML. However, a durable response is limited by resistance-conferring secondary kinase domain mutations, notably point mutations at D835. Therefore, there is a critical need for novel and potent treatments to eliminate AML cells harboring FLT3-D835 mutations. Here, we show the construction of TCR-engineered T cells targeting FLT3-D835 mutants, presented in the context of HLA-A *02:01. We first identified neoepitopes derived from D835Y/H/V/F/I with strong affinities to HLA-A *02:01 using NetMHCpan 4.1. These neoepitopes could form the stable complex with HLA-A *02:01 on the cell surface. Furthermore, CD8 + T cells specific for D835H were detected in the peripheral blood of AML patients carrying D835H mutant at different stages of the illness, most of which were memory T cells. Moreover, one neoepitope (Neo-D835H) induced IFN-γ secretion when co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from AML patients with D835H mutant. Neo-D835H-specific T cells were effectively generated through the rapid stimulation of CD8 + T cells isolated from an HLA-A *02:01-positive healthy donor with Neo-D835H-loaded autologous dendritic cells. These T cells were recognized by Neo-D835H-HLA-A2-Tetramer (0.37%) and consequently isolated by flow cytometry to perform TCRα and TCRβ sequencing. Both the TCRA and TCRB clonotypes showed dominant sequences that accounted for more than 50% of the total, suggesting that T cells with these dominant TCRA and TCRB-paired sequences were likely Neo-D835H-specific. We thus next generated TCR-engineered T cells with these TCRA- and TCRB-paired sequences for further functional analysis. These TCR-engineered T cells recognized Neo-D835H and showed cytotoxic activity against Neo-D835H-pulsed T2 cells in an antigen dose-dependent manner, while showed no responses to the T2 cells pulsed with the corresponding wild-type peptide. Taken together, we present novel neoantigens with promise as immunotherapy targets for AML and other hematologic malignancies with FLT3-D835 mutations. Future studies will evaluate the efficacy of Neo-D835H-specific TCR-engineered T-cell-based immunotherapy in vivo in patient-derived xenograft murine model and TCR-based therapeutic approaches targeting other FLT3-TKD mutations.

PHI-101 As a Potent Next-Generation FLT3 Inhibitor, Overcome Resistances in Previously Treated Patients with FLT3-ITD or TKD Acute Myeloid Leukemia: Results of a Phase Ia/Ib Clinical Trial

Background: The FLT3 receptor is mutated in about 30% of acute myeloid leukemia (AML) patients (pts) and has proven to be an attractive therapeutic target. The most prevalent alteration is internal tandem duplication (ITD) of the juxtamembrane domain, and several FLT3 inhibitors (e.g. gilteritinib, quizartinib, midostaurin) succeddfully target this mutation. However, patients frequently relapse due to resistance to these FLT3 inhibitors after initial clinical responses, often caused by the secondary mutations observed in the kinase domain (TKD) region. PHI-101, an orally bioavailable novel small molecule, is a next-generation FLT3 inhibitor that shows a potent anti-leukemic activity in primary AML samples harboring several FLT3-resistance mutations in preclinical studies. Global Phase I clinical trials with PHI-101 (NCT04842370) are currently underway in refractory or relapsed (R/R) AML pts. Study Design and Methods: The open-label, multicenter AML clinical trial consists of phase Ia, dose-escalation, and phase Ib, dose-expansion, utilizing oral PHI-101 tablets as a single-agent with R/R AML pts. If dose-escalation concludes with no dose-limiting toxicities (DLT), dose-expansion proceeds upon recommendation from the Safety Monitoring Committee (SMC). Response assessments with PHI-101 were reported according to 2017 ELN guidelines. The variant allelic frequency of mutations of FLT3 and other AML-related mutations was analyzed using NGS with bone marrow aspirates collected from pts before and after PHI-101 treatment. Blood samples were collected to evaluate pharmacokinetics (PK) and the plasma inhibitory assay (PIA) using FLT3-mutant cell lines. Together, these assessments were considered to determine the recommended dose for expansion (RDE) in phase Ib trials. Results: Phase Ia clinical trials of PHI-101 was designed to evaluate for safety and tolerability at five dose levels ranging from 40 mg to 200 mg. A total of 13 R/R AML pts were enrolled in the phase Ia and no DLTs were reported from daily doses of PHI-101 during the 28-day cycle. The target RDE at 160 mg was determined based on several considerations, including the PIA test achieving >85% inhibition of phosphor-FLT3, all available adverse events, PK data, and recommendation by the SMC. As of May 2023, with 6 pts enrolled in phase Ib, a total of 19 pts (5 FLT3-wild type (26.3%) and 14 FLT3 mutated (73.7%)), have been treated with PHI-101. Thirteen pts (68.4%) had more than 3 prior anti-leukemic treatments. Twelve of 13 pts (92%) assessed for the leukemic blast evaluation achieved clinical responses-4 pts with composite complete remissions [CRc, including complete remission with incomplete hematological recovery (CRi) and morphological leukemia free state (MLFS)] within 1 cycle of PHI-101 treatment. Ten pts were R/R following previous treatment with other FLT3 inhibitors (gilteritinib, quizartinib, midostaurin, or HM43239). 88 % of these pts achieved clinical responses including 3 CRc and 1 partial response (PR). Notably, all 5 R/R pts after gilteritinib treatment had clinical responses that included 2 with CRc. In addition, a patient with both FLT3-ITD and TKD (D835E and N676K) mutations who was resistant to 3 different FLT3 inhibitor treatments achieved PR and continued PHI-101 treatment for 6 cycles. Among pts who received 160 mg of PHI-101, 4 pts (57.1%) responded: 2 (28.6%) achieved CRi, 1(14.3%) achieved MLFS, and 1 (14.3%) achieved PR. Conclusion: In dose-escalating phase Ia clinical trials, PHI-101 was well tolerated at all dose levels with no DLTs. Phase Ib dose-expansion trials with 160 mg daily dosing are currently ongoing. PHI-101 has delivered CRcs at 120 mg and 160 mg. In this first-in-human, phase Ia/Ib trial of PHI-101, 92% of FLT3-wild or -mutated AML pts showed clinical responses even after having previously been treated with otherFLT3 inhibitors. More notably, 100% of pts who had relapsed after prior gilteritinib treatment showed clinical responses. The data from the clinical study of PHI-101to date demonstrates significant efficacy, particularly in R/R FLT3 mutant AML pts.

Concurrent PTEN and PDGFRB Alterations Characterize Storiform Collagenoma.

Storiform collagenoma is a rare mesenchymal skin tumor that is composed of thickened collagen bundles arranged in a characteristic storiform pattern with a relatively hypocellular CD34-positive spindle cell component. Storiform collagenoma is most often sporadic, but multiple lesions can occur in Cowden syndrome, which is characterized by germline alterations in PTEN (phosphatase and tensin homolog) on chromosome 10. Here, we investigated the molecular pathogenesis of storiform collagenoma using a targeted next-generation DNA sequencing platform, including 5 sporadic cases and one case associated with Cowden syndrome. Recurrent PTEN alterations were identified in all cases, with biallelic PTEN inactivation observed in the case associated with Cowden syndrome and one sporadic case. Unexpectedly, we also identified recurrent activating mutations in the platelet-derived growth factor receptor beta (PDGFRB) gene. This included a missense substitution in the D5 Ig-like domain of PDGFRB in the Cowden syndrome-associated case. In addition, we report missense alterations in the juxtamembrane domain of PDGFRB in 4 of 5 (80%) sporadic cases, including mutations that have been previously described in sporadic myofibroma and myopericytoma. Therefore, we confirm the neoplastic nature of storiform collagenoma, we expand the spectrum of reported PDGFRB alterations in mesenchymal tumors and we suggest a possible collaborative role for PTEN and PDGFRB in the pathogenesis of storiform collagenoma.

A Review of FLT3 Kinase Inhibitors in AML.

Acute myeloid leukemia (AML) is a highly aggressive illness distinguished by the accumulation of abnormal hematopoietic precursors in both the bone marrow and peripheral blood. The prevalence of FLT3 gene mutations is high and escalates the probability of relapse and mortality. The survival rates for AML patients, particularly those over 65, are low. FLT3 mutation screening at diagnosis is mandatory, and FLT3 inhibitors are crucial in treating AML patients with mutations. There are two categories of FLT3 mutations: FLT3-ITD located in the juxtamembrane domain and FLT3-TKD in the tyrosine kinase domain. FLT3-ITD is the most common type, affecting nearly a quarter of patients, whereas FLT3-TKD only affects 6-8% of patients. FLT3 inhibitors are now crucial in treating AML patients with FLT3 mutations. When dealing with FLT3-mutated AML, the recommended course of treatment typically involves chemotherapy and midostaurin, followed by allogeneic hematopoietic cell transplantation (HCT) to maximize the likelihood of success. Maintenance therapy can lower the risk of relapse, and gilteritinib is a better option than salvage chemotherapy for relapsed or refractory cases. Clinical trials for new or combined therapies are the most effective approach. This review discusses treatment options for patients with FLT3-mutated AML, including induction chemotherapy and options for relapsed or refractory disease. Additional treatment options may become available as more studies are conducted based on the patient's condition and susceptibility.

δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte-neuron cadherin interactions.

Astrocytes control the formation of specific synaptic circuits via cell adhesion and secreted molecules. Astrocyte synaptogenic functions are dependent on the establishment of their complex morphology. However, it is unknown if distinct neuronal cues differentially regulate astrocyte morphogenesis. δ-Catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. We found δ-catenin is also highly expressed by astrocytes and required both in astrocytes and neurons for astrocyte morphogenesis. δ-Catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and biochemical analyses to reveal that δ-catenin interacts with the N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

Targeting NANOG and FAK via Cx26-derived Cell-penetrating Peptides in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in preclinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

Golgi α-mannosidases regulate cell surface N-glycan type and ectodomain shedding of the transmembrane protease corin

Corin is a transmembrane protease that activates natriuretic peptides on the cell membrane. Reduced cell surface targeting or increased ectodomain shedding disrupts cell membrane homeostasis of corin, thereby impairing its cell surface expression and enzyme activity. N-glycans are essential in corin ectodomain shedding. Lack of N-glycans promotes corin ectodomain shedding in the juxtamembrane and frizzled-1 domains. The nascent N-glycans, transferred onto the polypeptide of corin, undergo multistep N-glycan processing in the endoplasmic reticulum and Golgi. It remains unclear how trimming by Golgi α-mannosidases, the critical N-glycan processing steps in N-glycan maturation, may regulate corin biosynthesis. In this study, we examined the effects of kifunensine and swainsonine, the inhibitors for α-mannosidases I and II, on corin expression and function. Western analysis of corin proteins in cell lysates and conditioned media from the inhibitor-treated corin-stable HEK293cells and AC16cells showed that both α-mannosidases I and II were required to maintain complex N-glycans on cell surface corin and protect corin from ectodomain shedding in the juxtamembrane and frizzled-1 domains. Cell viability analysis revealed that inhibition of α-mannosidase I or II sensitized cardiomyocytes to hydrogen peroxide-induced injury via regulating corin. Moreover, either one of the two coding genes was sufficient to perform Golgi α-mannosidase I trimming of N-glycans on corin. Similarly, this sufficiency was observed in Golgi α-mannosidase II-coding genes. Inhibition of ectodomain shedding restored corin zymogen activation from kifunensine- or swainsonine-induced reduction. Together, our results show the important roles of Golgi α-mannosidases in maintaining cell membrane homeostasis and biological activities of corin.

In silico targeting of colony-stimulating factor-1 receptor: delineating immunotherapy in cancer.

Delineate structure-based inhibition of colony-stimulating factor-1 receptor (CSF1R) by small molecule CSF1R inhibitors in clinical development for target identification and potential lead optimization in cancer therapeutics since CSF1R is a novel predictive biomarker for immunotherapy in cancer. Compounds were in silico modelled by induced fit docking protocol in a molecular operating environment (MOE, MOE.v.2015). The 3-dimensional (3D) X-ray crystallized structure of CSF1R kinase (Protein Databank, ID 4R7H) was obtained from Research Collaboratory for Structural Bioinformatics (RSCB) Protein Databank. The 3D conformers of edicotinib, DCC-3014, ARRY-382, BLZ-945, chiauranib, dovitinib, and sorafenib were obtained from PubChem Database. These structures were modelled in Amber10:EHT molecular force field, and quick prep application was used to correct and optimize the structures for missing residues, H-counts, termini capping, and alternates. The binding site was defined within the vicinity of the co-crystallized ligand of CSF1R kinase. The compounds were docked by the triangular matcher placement method and ranked by the London dG scoring function. The docked poses were further refined by the induced fit method. The pose with the lowest binding score (ΔG) was used to model the ligand interaction profile in Discovery Studio Visualizer v17.2. The co-crystallized ligand was docked in its apo conformation, and root-mean-square deviation was computed to validate the docking protocol. All 7 CSF1R inhibitors interact with residue Met637 exhibiting selectivity except for edicotinib. The inhibitors maintain CSF1R in an auto-inhibitory conformation by interacting with Asp797 of the Asp-Phe-Gly (DFG) motif and/or hindering the conserved salt bridge formed between Glu633 and Lys616 thus stabilizing the activation loop, or interacting with tryptophan residue (Trp550) in the juxtamembrane domain. DCC-3014, ARRY-382, BLZ-945, and sorafenib bind with the lowest binding energy with CSF1R kinase. Pyrimidines are potent inhibitors that interact with CSF1R residues. DCC-3014 and ARRY-382 exhibit exceptional pharmaceutical potential exhibiting great structural stability and affinity.

Bioinformatic analysis of KIT juxtamembrane domain mutations in Syrian GIST patients: jigsaw puzzle completed

BackgroundThe huge number of detected somatic KIT mutations highlights the necessity of in silico analyses that are almost absent in the relevant medical literature. The aim of this study is to report the mutation spectrum analysis of exon 11 encoding the juxtamembrane (JM) domain of the KIT gene in a group of Syrian GIST patients.MethodsForty-eight formalin-fixed paraffin-embedded GIST tissue samples, collected between 2006 and 2016, were retrieved from the pathological archives and analyzed for KIT exon 11 mutations by DNA sequencing. Structural/functional impact of detected variants was predicted using several bioinformatic tools.ResultsTwenty-one different variants have been detected in intron 10, exon 11, and intron 11 of the KIT gene, eight of which were novel changes. Mutations in exon 11 of the KIT gene were detected in 28 of 48 (58.3%) GIST patients and predicted to be pathogenic and cancer promoting. Specifically, age above 60 was very significantly associated with the negative selection of deletion mutations (p = .007), a phenomenon that points to deletion severity.ConclusionsSix bioinformatic tools have proved efficient in predicting the impact of detected KIT variations in view of published structural, experimental, and clinical findings.

How ITD Insertion Sites Orchestrate the Biology and Disease of FLT3-ITD-Mutated Acute Myeloid Leukemia.

Mutations of the FLT3 gene are among the most common genetic aberrations detected in AML and occur mainly as internal tandem duplications (FLT3-ITD). However, the specific sites of FLT3-ITD insertion within FLT3 show marked heterogeneity regarding both biological and clinical features. In contrast to the common assumption that ITD insertion sites (IS) are restricted to the juxtamembrane domain (JMD) of FLT3, 30% of FLT3-ITD mutations insert at the non-JMD level, thereby integrating into various segments of the tyrosine kinase subdomain 1 (TKD1). ITDs inserted within TKD1 have been shown to be associated with inferior complete remission rates as well as shorter relapse-free and overall survival. Furthermore, resistance to chemotherapy and tyrosine kinase inhibition (TKI) is linked to non-JMD IS. Although FLT3-ITD mutations in general are already recognized as a negative prognostic marker in currently used risk stratification guidelines, the even worse prognostic impact of non-JMD-inserting FLT3-ITD has not yet been particularly considered. Recently, the molecular and biological assessment of TKI resistance highlighted the pivotal role of activated WEE1 kinase in non-JMD-inserting ITDs. Overcoming therapy resistance in non-JMD FLT3-ITD-mutated AML may lead to more effective genotype- and patient-specific treatment approaches.

Mutation spectrum of FLT3 and significance of non-canonical FLT3 mutations in haematological malignancy.

Fms-like tyrosine kinase 3 (FLT3) is frequently mutated in haematological malignancies. Although canonical FLT3 mutations including internal tandem duplications (ITDs) and tyrosine kinase domains (TKDs) have been extensively studied, little is known about the clinical significance of non-canonical FLT3 mutations. Here, we first profiled the spectrum of FLT3 mutations in 869 consecutively newly diagnosed acute myeloid leukaemia (AML), myelodysplastic syndrome and acute lymphoblastic leukaemia patients. Our results showed four types of non-canonical FLT3 mutations depending on the affected protein structure: namely non-canonical point mutations (NCPMs) (19.2%), deletion (0.7%), frameshift (0.8%) and ITD outside the juxtamembrane domain (JMD) and TKD1 regions (0.5%). Furthermore, we found that the survival of patients with high-frequency (>1%) FLT3-NCPM in AML was comparable to those with canonical TKD. In vitro studies using seven representative FLT3-deletion or frameshift mutant constructs showed that the deletion mutants of TKD1 and the FLT3-ITD mutant of TKD2 had significantly higher kinase activity than wild-type FLT3, whereas the deletion mutants of JMD had phosphorylation levels comparable with wild-type FLT3. All tested deletion mutations and ITD were sensitive to AC220 and sorafenib. Collectively, these data enrich our understanding of FLT3 non-canonical mutations in haematological malignancies. Our results may also facilitate prognostic stratification and targeted therapy of AML with FLT3 non-canonical mutations.

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane ‘accessory’ proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.

Abstract LB214: A first-in-class bispecific antibody-drug conjugate (DM002) targeting HER3 and the juxtamembrane domain of MUC1

Abstract Despite the entry of new anti-cancer drugs into the market, there were more than 10 million deaths from cancer globally in 2021, with cancers of the lung, stomach, breast, and pancreas contributing to the most cancer-related deaths in China and the USA. Accordingly, there is an urgent need for improved therapeutic interventions. Antibody-drug conjugates (ADC) are novel drugs that exploit the specificity of a monoclonal antibody for target antigens expressed on cancer cells in order to achieve targeted delivery of a potent cytotoxic payload. More recently, bispecific ADCs (BsADC) targeting two tumor-associated antigens (TAA) have been developed to further amplify tumor cell specificity while minimizing toxicity to normal tissue, thus allowing for broader therapeutic windows. HER3 and MUC1 are two TAAs that are commonly expressed/co-expressed on multiple tumor types, including lung, gastric, breast and pancreatic cancer; however, neither antigen has been successfully targeted by effective drugs: of the candidates targeting HER3 antigen being evaluated in human clinical studies, most are well-tolerated, but with limited efficacy. On the other hand, high levels of MUC1 in diseased tissues can undergo auto-proteolysis, so that drugs targeting the MUC1-N region will be neutralized before reaching the tumor tissues, thereby limiting the recognition of tumor cells. To overcome these challenges, we generated fully human bispecific antibody candidates with human-monkey cross-species reactivity that target HER3 and the juxtamembrane domain of MUC1. These bsAbs exhibit higher endocytic activity in HER3-low tumor cell lines compared with other currently available HER3 mAb. These bsAbs were subsequently conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker to obtain first-in-class BsADC candidates, DM002. DM002 candidates showed robust anti-tumor activity in multiple CDX and PDX models of lung, breast, gastric and pancreatic cancer; most notably, DM002 candidates outperformed benchmark ADCs in BP0508 lung PDX models. Together, these data indicate that DM002 will be a promising therapeutic drug for patients with HER3 and MUC1 co-expressing tumors. Citation Format: Yifu Zhang, Chenzhang Shang, Nannan Wang, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. A first-in-class bispecific antibody-drug conjugate (DM002) targeting HER3 and the juxtamembrane domain of MUC1 [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 LB214.

Abstract 8: Differential flt3 mutational status in acute myeloid leukemia predicts sensitivity to flt3 inhibitor gilteritinib in vitro and in vivo

Abstract Acute Myeloid Leukemia (AML) is a heterogenous hematopoietic cancer characterized by abnormal differentiation and uncontrolled proliferation of hematopoietic progenitor cells. Recent approaches to treatment, which aim to improve outcomes in refractory AML, include strategies to molecularly target specific receptor-mediated signaling pathways which regulate cell growth and proliferation. One target-specific approach is the inhibition of fms-like tyrosine kinase 3 (FLT3). FLT3 is expressed on the surface of hematopoietic progenitor cells and plays an important role in regulating proliferation, survival, and differentiation of multipotent stem cells. The presence of internal tandem duplications (ITDs) at the juxta membrane domain within the FLT3 receptor are common driver mutations in 30% of AML patients. In this study, we utilized 3 luciferase-expressing human AML models, MV4-11, MOLM-13, and, OCI-AML3 to determine the prognostic impact of FLT3 inhibitor Gilteritinib on the allelic ratio of ITD versus wildtype allele. OCI-AML3 expresses wild-type FLT3 whereas MOLM-13 is heterozygous for FLT3-ITD mutations, and MV4-11 possesses a homozygous in-frame tandem repeat insertion in the FLT3 allele. Here we demonstrate that FLT3-ITD mutational status impacts the efficacy of Gilteritinib in both in vitro and in vivo AML models. The in vitro assays showed that treatment with Gilteritinib resulted in significant dose dependent reduction in overall cell viability and bioluminescence in the MV4-11 cell line, while MOLM-13 was only moderately sensitive, and OCI-AML3 was least sensitive to Gilteritinib treatment. Additionally, we evaluated the downstream MAPK and PI3K signaling pathways as FLT3-ITD oncogenic mutations result in ligand-independent constitutive activation of signaling pathways mediating leukemic transformation. For in vivo validation, MV4-11, MOLM-13, and OCI-AML3 luciferase expressing cells were implanted systemically into NCG mice and once tumors were established, we evaluated the survival benefit in response to Gilteritinib and various standard of care agents. In accordance with our in vitro findings, the quantification of tumor progression by bioluminescent imaging showed the highest therapeutic activity of Gilteritinib in MV4-11 tumor bearing mice through selective targeting of FLT3-ITD mutations. Taken together, our in vitro and in vivo results demonstrate that differential FLT3-ITD mutational status in AML models impacts Gilteritinib treatment response and downstream signaling events with the highest activity in MV4-11 followed by MOLM-13, and lack of activity in OCI-AML3 harboring wild-type FLT3. Citation Format: Bincy John, Pasupathi Sundaramoorthy, Elizabeth Rainbolt, Andrew Wong, Zachary Ward, Justin Avery, Savannah Todd, Shasta Kidder, Diana Gietl, Chassidy Hall, David Harris. Differential flt3 mutational status in acute myeloid leukemia predicts sensitivity to flt3 inhibitor gilteritinib in vitro and in vivo [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 8.

Abstract 3877: Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer

Abstract The estrogen receptor-positive (ER+) subtype accounts for 70% of breast cancer, and endocrine therapies that target ER are effective in the early-stage clinical setting. However, endocrine resistance is ubiquitous in late-stage metastatic breast cancer (MBC). The chief mechanism is the acquisition of ER mutations (ERmuts) in 25% of endocrine-resistant tumors; ERmuts are strongly resistant to aromatase inhibitor (AI) endocrine therapy. We previously identified four novel acquired activating mutations in human epidermal growth factor receptor 2 (HER2) by whole exome sequencing (WES) of paired ER+ MBC in a clinical cohort. These HER2 mutations (HER2muts) were enriched in multiple resistant tumors, and like ERmuts, were resistant to AIs. In contrast to ERmuts, these HER2muts also conferred complete resistance to all other classes of endocrine therapy, as well as cross-resistance to combination therapy with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors; the latter is standard-of-care in the refractory setting in ER+ MBC. At the same time, these HER2muts had variable responses to HER2-targeting therapies used in HER2+ breast cancer. Thus, HER2muts represent only the second validated mechanism of clinical endocrine resistance in ER+ MBC, accounting for ~8% of cases, and that is therapeutically intractable. Systematic phenotypic characterization of HER2muts in ER+ MBC is a prerequisite for effective targeting of this class of resistance. We used cBioPortal to identify >20 novel HER2 variants of unknown significance (VUS) that were frequent pan-cancer, in ER+ breast tumors, or annotated in a domain-restricted HER2 saturation mutagenesis screen, and that had not been previously characterized in ER+ MBC. We also identified a novel HER2 VUS by WES in an ER+/HER2+ tumor with resistance to HER2 therapy, from a clinical cohort of breast cancer patients. We phenotypically characterized all VUS through clinically comprehensive targeted therapy viability assays, and downstream activation assays. In sum, we identified seven novel activating HER2muts, spanning domains, with clinically important phenotypes in ER+ MBC. We found these HER2muts also conferred complete resistance to all classes of endocrine therapies, and cross-resistance to CDK4/6 inhibition. While novel HER2muts had variable responses to HER2-targeting therapies, they could be endocrine re-sensitized by combination with an irreversible tyrosine kinase inhibitor. Lastly, we report that constitutive HER2 homodimerization via reduction-sensitive disulfide bridges that occur as a consequence of mutations in the extracellular and juxtamembrane domains is a more common mechanism of HER2 hyperactivation than previously appreciated. This finding has unique functional consequences, and provides important structure-function insight into HER2 biology, beyond its immediate clinical ramifications in ER+ MBC. Citation Format: Nelson Vicente Guevara, Zirui Zhu, Esha Jain, Jennifer Kavran, Selime Arslan, Nikhil Wagle, Utthara Nayar. Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer. [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 3877.

Abstract 3173: Integrative structural models of oncogenic mutants within the trans- and juxta-membrane region of human epidermal growth factor receptors (HERs)

Abstract The trans- and juxtamembrane domains (TMD/JMD) of human epidermal growth factor receptors (HER/EGFR/ErbB) regulate the helix-helix dimerization interface to control receptor tyrosine kinase (RTK) activation. Patient-identified non-small cell lung cancer (NSCLC) mutations occur all along the EGFR (HER1) and HER2 TMD and JMD, but our structural understanding of how these patient-derived TMD/JMD mutations hijack wild-type RTK function to drive cancer is lacking. In this work, we integrate tools in experimental biophysics and computational modeling to evaluate the structural mechanism(s) driving these interactions in atomic detail. We use nuclear magnetic resonance (NMR) spectroscopy to assess how oncogenic mutations within the TMD/JMD perturb the architecture/interactions of the dimer interface. NMR-derived models are used to seed molecular dynamics (MD) simulations to probe how mutations affect TMD association and activation/stabilization via dimerization in a realistic membrane environment. We apply this strategy and focus our initial efforts on the EGFRA647T variant. We computed the free energy landscape from our simulations and show that the active state for the EGFRA647T-HER2WT heterodimer is stabilized by 10 kcal/mol. Conversely, the wild-type EGFR-HER2 heterodimer is stabilized by 5 kcal/mol for the inactive state suggesting the propensity of the EGFRA647T mutant favors the active state. These results are part of a multidisciplinary collaboration that share the common goal of constructing holistic models that detail how TMD/JMD variants are aberrantly activated in cancer. Citation Format: Christina Mercado, Abdullah Al Mamun, Younghee Park, Christine M. Lovly, Jens Meiler, Kaitlyn Ledwitch. Integrative structural models of oncogenic mutants within the trans- and juxta-membrane region of human epidermal growth factor receptors (HERs) [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 3173.

Abstract 3393: A novel targetable G3BP stress granule assembly factor 1 (G3BP1)-colony stimulating factor 1 receptor (CSF1R) fusion in acute megakaryoblastic leukemia (AMKL)

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease, and targeted therapy has been shown to improve the outcomes of AML patients. Recently, we identified a novel G3BP stress granule assembly factor 1 (G3BP1)-colony stimulating factor 1 receptor (CSF1R) fusion gene by RNA sequencing in a patient with acute megakaryoblastic leukemia (AMKL). G3BP1-CSF1R (GC) juxtaposed exon 8 of G3BP1 to exon 12 of CSF1R and was predicted to encode a fusion protein containing the disrupted juxtamembrane domain (JMD) and the intact kinase domain of CSF1R. A similar CSF1R rearrangement has been reported in an AMKL cell line, MKPL1, which harbors the RBM6-CSF1R fusion. As the second CSF1R fusion discovered in AMKL, it remains unclear whether GC is a novel oncogenic driver and whether it is involved in dictating megakaryocytic phenotype. The aim of this study was to characterize the GC fusion and to investigate its role in AMKL pathogenesis and its targetability. Immunofluorescence studies showed that GC and G3BP1 were located in the cytoplasm, unlike the wide-type CSF1R which exhibited vesicular localization. Co-IP studies indicated that GC could exist as dimers in the absence of ligand stimulation. Two cytokine-dependent cell lines (TF1 and M07E) transduced with GC lentivirus showed cytokine-independent growth and constitutive activation of CSF1R and its downstream signaling, indicating the transforming ability of GC. Consistently, NSG mice transplanted with GC-expressing TF1 cells developed AML. In vitro drug studies revealed that GC-transformed TF1 (TF1-GC) and MKPL1 cells were sensitive to dasatinib and pexidartinib, as demonstrated by inhibition of kinase activation, reduced cell proliferation, and increased apoptosis. Moreover, in NSG mice transplanted with TF1-GC cells, dasatinib and pexidartinib significantly decreased leukemia burden and prolonged survival. Using flow cytometry, we observed that GC increased cell size, granularity, and megakaryocytic markers (CD41 and CD61) in M07E, TF1, and human cord blood CD34+ stem and progenitor cells, suggesting that GC could promote megakaryocytic skewing. Taken together, we identified and characterized a novel targetable GC fusion in AMKL. These findings may provide insights into the role of CSF1R in AMKL pathogenesis and a rationale for CSF1R-targeted therapy in AML patients with CSF1R activating alterations. Citation Format: Xi Luo, Chi Keung Cheng, Hoi Yun Chan, Yuet Fong Kam, Kam Tong Leung, Chi Kong Li, Margaret Heung-Ling Ng. A novel targetable G3BP stress granule assembly factor 1 (G3BP1)-colony stimulating factor 1 receptor (CSF1R) fusion in acute megakaryoblastic leukemia (AMKL) [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 3393.

Abstract 2412: MET inhibition enhances the effect of radiation in MET dysregulated non-small cell lung cancer models

Abstract Objective: Alterations in the MET receptor are seen in up to 10% of patients with non-small cell lung cancer (NSCLC). A mutation in exon 14, which results in deletion of the intracellular juxtamembrane domain of the receptor, leading to enhanced signaling, is the most common MET mutation in NSCLC. MET is involved in multiple pathways associated with apoptosis and radiation response. We investigated the effects of inhibiting MET with capmatinib, a potent and clinically relevant ATP-competitive kinase inhibitor, in combination with radiation in MET-dependentNSCLC models. Methods: MET exon 14 mutated and MET amplified NSCLC cell lines treated with radiation or capmatinib were assessed with proliferation, clonogenic survival, apoptosis, and DNA damage signaling and repair assays. MET signaling proteins were evaluated by Western blotting. In vivo growth responses to the MET inhibitor capmatinib with and without radiation were evaluated in patient derived xenograft (PDX) models established from both MET amplified and MET exon 14 skipping mutation containing patient tumors. Radiation was delivered in 10 daily fractions of 2 Gy and capmatinib was administered by oral gavage daily 1 hour prior to radiation at a dose 20 mg/kg for capmatinib. Immunohistochemistry (IHC) was performed to evaluate MET signaling and proliferation. Results: Radiation increased the expression and capmatinib inhibited phospho-AKT, phospho-s6,phospho-ERK, and phospho-STAT3 in our MET-driven NSCLC cell lines. Combination of capmatinib and radiation in clonogenic assays increased sensitivity to radiation in MET dysregulated cell lines. No radiation-enhancing effect was observed in MET wild-type and human bronchial epithelial cell lines. Campatinib alone significantly inhibited tumor growth in both ourMET exon 14 mutated and MET amplified xenograft models (p<0.01). Combination of capmatinib and radiation significantly delayed growth compared to vehicle control, capmatinib alone or radiation alone (p<0.01). IHC demonstrated inhibition of phospho-MET and phospho-S6 and a decrease in Ki67 with inhibition of MET. A significant decrease Ki67 proliferation was observed in the combination arm compared to radiation along or drug alone. Conclusion:Inhibition of MET enhanced the effect of radiation in multiple MET-altered NSCLC PDX models.Additional studies are currently underway evaluating the mechanisms of radiation sensitization with a focus on MET-induced apoptosis. Citation Format: Shrey K. Ramesh, Saahil N. Javeri, Kwangok P. Nickel, Ahmet Cifci, Rachel L. Minne, Randall J. Kimple, Andrew M. Baschnagel. MET inhibition enhances the effect of radiation in MET dysregulated non-small cell lung cancer 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 2412.

Efficient sampling of TrkA transmembrane domain dimerization captures functionally distinct structural ensembles.

Upon ligand binding, the transmembrane domains (TMD) of the receptor tyrosine kinase (RTK) dimerize, induce conformational change in the juxtamembrane domain (JMD), and trigger downstream phosphorylation that eventually determines fate of the cell. To overcome the experimental difficulty in probing the TMD dimerization events in membrane bilayers, we employed all-atom molecular dynamics with a novel membrane mimetic solvent Simple Carbon Solvent Ethane (SCSE) to efficiently sample the TMD dimerization processes for tropomyosin receptor kinase A (TrkA). Spontaneous and stable TMD dimers were observed in less than 50 ns of simulation time from an initially separated grid configuration. We then performed hierarchical clustering using a customized distance metric based on TMD contact residues, and revealed major structural ensembles of TMD dimers. We demonstrated the validity of the resolved TMD dimer structures by performing MM-GBSA free energy calculation and molecular dynamics with full-tail lipid bilayers. As the residues in contact in the most populated TMD dimer ensemble from our SCSE simulations overlap with those found to be critical for kinase activation, we hypothesized that the captured TMD dimer corresponds to the active state, while the previously characterized NMR structure is the inactive state. To understand the role of different TMD dimer conformations in downstream signaling, we assembled TMD+JMD constructs using the captured in simulation dimer structure and the NMR structure. We modelled the system with Highly Mobile Membrane Mimetic (HMMM) for accelerated peptide-lipid interactions and found that the captured TMD dimer leads to JMD conformations distinct from those tethered to the NMR dimer structure, which may enable the kinase domain for cross-phosphorylation. Our method represents a new approach to study RTK dimerization and sample physiologically relevant dimer structures.