Juxtamembrane Domain Mutations Research Articles

Pharmaco-phosphoproteomic analysis of cancer-associated KIT mutations D816V and V560G.

The CD117 mast/stem cell growth factor receptor tyrosine kinase (KIT) is critical for haematopoiesis, melanogenesis and stem cell maintenance. KIT is commonly activated by mutation in cancers including acute myeloid leukaemia, melanoma and gastrointestinal stromal tumours (GISTs). The kinase and the juxtamembrane domains of KIT are mutation hotspots; with the kinase domain mutation D816V common in leukaemia and the juxtamembrane domain mutation V560G common in GISTs. Given the importance of mutant KIT signalling in cancer, we have conducted a proteomic and phosphoproteomic analysis of myeloid progenitor cells expressing D816V- and V560G-KIT mutants, using an FDCP1 isogenic cell line model. Proteomic analysis revealed increased abundance of proteases and growth signalling proteins in KIT-mutant cells compared to empty vector (EV) controls. Pathway analysis identified increased oxidative phosphorylation in D816V- and V560G-mutant KIT cells, which was targetable using the inhibitor IACS010759. Dysregulation of RNA metabolism and cytoskeleton/adhesion pathways was identified in both the proteome and phosphoproteome of KIT-mutant cells. Phosphoproteome analysis further revealed active kinases such as EGFR, ERK and PKC, which were targetable using pharmacological inhibitors. This study provides a pharmaco-phosphoproteomic profile of D816V- and V560G-mutant KIT cells, which reveals novel therapeutic strategies that may be applicable to a range of cancers.

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.

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.

Abstract P4-01-02: A spectrum of secondary mutations in HER2 augment breast cancer cell growth and reduce neratinib sensitivity in HER2-mutant breast cancer

Abstract Activating mutations in HER2 (ERBB2) drive tumor growth and antiestrogen resistance in ~5% of metastatic ER+ breast cancers. Treatment with the irreversible HER2 tyrosine kinase inhibitor neratinib, in combination with fulvestrant, leads to objective responses in patients with HER2-mutant ER+ breast cancer. However, acquired resistance is common. The majority of HER2-mutant tumors progressing on neratinib-based therapy acquire secondary mutations in HER2. Except for the HER2T798I gatekeeper mutation, whether these secondary HER2 mutations are causal to neratinib resistance is not known. We hypothesized that secondary HER2 mutations augment HER2 pathway activation and promote neratinib resistance. We first studied acquired secondary HER2 mutations found in circulating tumor DNA (ctDNA) of 2 patients with breast cancer harboring the primary HER2L869R hotspot mutation. The following mutations were absent at baseline but detected in ctDNA at the time of progression on neratinib: S310Y, L755S, D769Y, and T798I (patient 1, partial response to neratinib/fulvestrant >16 months); and T862A, T798I, S310F, and I767M (patient 2, stable disease >7 months). We stably expressed each mutation alone or in combination with HER2L869R in MCF10A breast epithelial cells. All single mutations, with the exception of T862A and T798I, promoted growth factor-independent proliferation and growth in 3D Matrigel. Enhanced growth was blocked by treatment with 10 nM neratinib. Cells expressing HER2L869R together with S310F, I767M, and D769Y remained highly sensitive to neratinib (IC50 <6 nM). In contrast, HER2L869R/L755S and HER2L869R/T862A (double-mutant) cells formed large acini in 3D Matrigel in the presence of neratinib, and exhibited a 3- to 5-fold higher IC50 than HER2L869R in 2D cell viability assays. Western blot analysis revealed that HER2L869R/L755S and HER2L869R/T862A cells required a higher concentration of neratinib (50-100 nM; 4 h) to block phosphorylation of HER2 and downstream targets AKT, ERK, and S6 relative to cells transduced with HER2L869R alone (10 nM neratinib). While HER2T862A alone did not increase HER2 pathway activation or growth factor-independent growth compared to wild type (WT) HER2, phosphorylation of HER2, AKT, ERK, and S6 was elevated in cells expressing HER2L869R/T862A relative to each single-mutant. Likewise, cells expressing HER2L869R/T862A formed larger and more invasive acini in 3D Matrigel. Computational modeling predicted that the active state of HER2 was stabilized in the HER2L869R/T862A double-mutant relative to that of either single mutant. The overall energetic barrier for the active to inactive state transition also increased for the double mutant compared to that of each single mutant. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) simulations showed that neratinib binding affinity is higher for HER2L869R/T862Arelative to HER2L869R or HER2WT. Two secondary HER2 juxtamembrane domain mutations, E698del and P699L, were also acquired in HER2V777L-driven breast cancers that had progressed on neratinib. However, MCF10A cells co-transduced with V777L/E698del and with V777L/9669L remained highly sensitive to neratinib. Conclusions: In tumors with an oncogenic primary HER2 mutation, acquired secondary mutations in HER2L755S and HER2T862A reduced neratinib sensitivity. The HER2L869R/T862A double-mutant stabilizes the HER2 activation state and enhances HER2 signaling output which, in turn, was incompletely blocked by neratinib. Some but not all acquired secondary HER2 mutations enhance breast tumor growth and/or are causal to neratinib resistance. Citation Format: Arnaldo Andres Marin, Abdullah Al Mamun, Hiroaki Akamatsu, Dan Ye, Dhivya Sudhan, Benjamin Brown, Monica Red Brewer, Lisa Eli, Jens Meiler, Carlos L Arteaga, Ariella B Hanker. A spectrum of secondary mutations in HER2 augment breast cancer cell growth and reduce neratinib sensitivity in HER2-mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-02.

FLT3-TKD in the Prognosis of Patients With Acute Myeloid Leukemia: A Meta-Analysis

Background: Fms-like tyrosine kinase 3 (FLT3) gene mutations occur in approximately 30% of all AML patients. Internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain (TKD) are two distinct types of FLT3 mutations. FLT3-ITD has been determined as an independent poor prognostic factor, but the prognostic impact of FLT3-TKD remains controversial. Hence, we performed a meta-analysis to investigate the prognostic significance of FLT3-TKD in patients with AML. Methods: A systematic retrieval of studies on FLT3-TKD in patients with AML was performed in PubMed, Embase, and CNKI databases on Sept. 30th, 2020. Hazard ratio (HR) and its 95% confidence intervals (95% CIs) were used to determine the effect size. Meta-regression model and subgroup analysis were used for heterogeneity analysis. Begg’s and Egger’s tests were performed to detect potential publication bias. The sensitivity analysis was performed to evaluate the stability of findings in meta-analysis. Findings: Twenty prospective cohort studies (n=10970) on the prognostic effect of FLT3-TKD in AML were included: 9744 subjects with FLT3-WT and 1226 subjects with FLT3-TKD. We found that FLT3-TKD revealed no significant effect on DFS (HR=1.12, 95% CI: 0.90-1.41) and OS (HR=0.98, 95% CI: 0.76-1.27) in general. However, meta-regressions demonstrated that ethnicity contributed to the high heterogeneity observed in the prognosis of FLT3-TKD in AML. To be specific, FLT3-TKD represented a beneficial prognosis of DFS (HR=0.56, 95% CI: 0.37-0.85) and OS (HR=0.63, 95% CI: 0.42-0.95) for Asians while it represented an adverse prognosis of DFS for Caucasians with AML (HR=1.34, 95% CI: 1.07-1.67). Interpretation: Our results showed that FLT3-TKD revealed no significant effects on DFS and OS of patients with AML, which is consistent with the controversial status nowadays. Distinct prognosis of FLT3-TKD in AML between Asians and Caucasians may attribute to different chemotherapy regimens (including the dose of cytarabine and daunorubicin in conventional 3+7 induction and the clinical usage of HHT) and diverse genetic background. The dose of cytarabine (100 mg/m2) and daunorubicin (50 mg/m2) used in Asian countries in 3+7 induction therapy may benefit Caucasians with AML carrying FLT3-TKD. Funding Information: National Natural Science Foundation of China (NSFC, Grant No. 82000150). Declaration of Interests: We declare no competing interests.

A Molecular Test for Quantifying Functional Notch Signaling Pathway Activity in Human Cancer.

Simple SummaryThe Notch signal transduction pathway is important for various physiological processes, including immune responses, and plays a role in many diseases, for example cancer. We have developed a new assay to quantitatively measure Notch pathway activity, and we validated it using data from various human cancer cell lines. The assay can be applied across different cell types, and offers numerous possibilities to explore the contribution of the Notch pathway to tumor formation and the stratification of cancer patients. We assessed Notch pathway activity in a cohort of T cell acute lymphoblastic leukemia (T-ALL) patient samples, and found that the pathway activity score more accurately reflects Notch pathway activity than a prediction on the basis of NOTCH1 mutations alone. Finally, we found that patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients who had T-ALL cells with higher activity.Background: The Notch signal transduction pathway is pivotal for various physiological processes, including immune responses, and has been implicated in the pathogenesis of many diseases. The effectiveness of various targeted Notch pathway inhibitors may vary due to variabilities in Notch pathway activity among individual patients. The quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. Methods: We here describe a new assay that infers a quantitative Notch pathway activity score from the mRNA levels of generally conserved direct NOTCH target genes. Following the calibration and biological validation of our Notch pathway activity model over a wide spectrum of human cancer types, we assessed Notch pathway activity in a cohort of T-ALL patient samples and related it to biological and clinical parameters, including outcome. Results: We developed an assay using 18 select direct target genes and high-grade serous ovarian cancer for calibration. For validation, seven independent human datasets (mostly cancer series) were used to quantify Notch activity in agreement with expectations. For T-ALL, the median Notch pathway activity was highest for samples with strong NOTCH1-activating mutations, and T-ALL patients of the TLX subtype generally had the highest levels of Notch pathway activity. We observed a significant relationship between ICN1 levels and the absence/presence of NOTCH1-activating mutations with Notch pathway activity scores. Patients with the lowest Notch activity scores had the shortest event-free survival compared to other patients. Conclusions: High Notch pathway activity was not limited to T-ALL samples harboring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate Notch signaling. The measured Notch pathway activity was related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than when it is predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.

FLT3 inhibitors in the treatment of acute myeloid leukemia: current status and future perspectives.

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene arise in 25-30% of all acute myeloid leukemia (AML) patients. These mutations lead to constitutive activation of the protein product and are divided in two broad types: internal tandem duplication (ITD) of the juxtamembrane domain (25% of cases) and point mutations in the tyrosine kinase domain (TKD). Patients with FLT3 ITD mutations have a high relapse risk and inferior cure rates, whereas the role of FLT3 TKD mutations still remains to be clarified. Additionally, growing research indicates that FLT3 status evolves through a disease continuum (clonal evolution), where AML cases can acquire FLT3 mutations at relapse - not present in the moment of diagnosis. Several FLT3 inhibitors have been tested in patients with FLT3-mutated AML. These drugs exhibit different kinase inhibitory profiles, pharmacokinetics and adverse events. First-generation multi-kinase inhibitors (sorafenib, midostaurin, lestaurtinib) are characterized by a broad-spectrum of drug targets, whereas second-generation inhibitors (quizartinib, crenolanib, gilteritinib) show more potent and specific FLT3 inhibition, and are thereby accompanied by less toxic effects. Notwithstanding, all FLT3 inhibitors face primary and acquired mechanisms of resistance, and therefore the combinations with other drugs (standard chemotherapy, hypomethylating agents, checkpoint inhibitors) and its application in different clinical settings (upfront therapy, maintenance, relapsed or refractory disease) are under study in a myriad of clinical trials. This review focuses on the role of FLT3 mutations in AML, pharmacological features of FLT3 inhibitors, known mechanisms of drug resistance and accumulated evidence for the use of FLT3 inhibitors in different clinical settings.

FLT3 mutations in acute myeloid leukemia: Therapeutic paradigm beyond inhibitor development.

FMS‐like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that plays an important role in hematopoietic cell survival, proliferation and differentiation. The most clinically important point is that mutation of the FLT3 gene is the most frequent genetic alteration and a poor prognostic factor in acute myeloid leukemia (AML) patients. There are two major types of FLT3 mutations: internal tandem duplication mutations in the juxtamembrane domain (FLT3‐ITD) and point mutations or deletion in the tyrosine kinase domain (FLT3‐TKD). Both mutant FLT3 molecules are activated through ligand‐independent dimerization and trans‐phosphorylation. Mutant FLT3 induces the activation of multiple intracellular signaling pathways, mainly STAT5, MAPK and AKT signals, leading to cell proliferation and anti–apoptosis. Because high‐dose chemotherapy and allogeneic hematopoietic stem cell transplantation cannot sufficiently improve the prognosis, clinical development of FLT3 kinase inhibitors expected. Although several FLT3 inhibitors have been developed, it takes more than 20 years from the first identification of FLT3 mutations until FLT3 inhibitors become clinically available for AML patients with FLT3 mutations. To date, three FLT3 inhibitors have been clinically approved as monotherapy or combination therapy with conventional chemotherapeutic agents in Japan and/or Europe and United states. However, several mechanisms of resistance to FLT3 inhibitors have already become apparent during their clinical trials. The resistance mechanisms are complex and emerging resistant clones are heterogenous. Further basic and clinical studies are required to establish the best therapeutic strategy for AML patients with FLT3 mutations.

Discovery & Characterization of Therapeutically Targetable Ntrk Point Mutations in Leukemia

Discovery & Characterization of Therapeutically Targetable Ntrk Point Mutations in Leukemia

A Test to Quantify NOTCH Pathway Activity in T Cell Acute Lymphoblastic Leukemia Patients

Background The NOTCH signaling pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis in many diseases including T-cell acute lymphoblastic leukemia (T-ALL). Over 70% of T-ALL patient samples contain mutations in NOTCH1 and/or FBXW7 that result in the activation of the NOTCH pathway. Various targeted drugs are available that inhibit NOTCH signaling, but their effectiveness varies due to variable NOTCH pathway activities among individual patients. Moreover, patients' leukemic cells that lack these mutations may still require NOTCH signaling. A quick and robust quantification of NOTCH pathway activity in primary patient samples would identify patients who could benefit from NOTCH targeted treatment. Aims In primary human T-ALL samples, we aimed to determine the NOTCH pathway activity in relation to active, intracellular NOTCH1 (ICN1) levels and in relation to NOTCH1 and/or FBXW7 mutations. Additionally, we investigated whether the NOTCH pathway activity score is more accurate than a mutation-based activity prediction. Methods Our test to assess functional NOTCH pathway activity in various cell types was applied to primary human T-ALL samples. The NOTCH test infers a quantitative NOTCH pathway activity score from mRNA levels of conserved direct NOTCH target genes based on a Bayesian network model. This model describes the causal relation between up- or downregulation of NOTCH target genes and the presence of an active or inactive NOTCH transcription complex. The Bayesian model was calibrated on publically available Affymetrix U133 Plus2.0 microarray datasets of samples with an active or inactive NOTCH pathway. Following validation on multiple cell types and malignancies, we scored NOTCH pathway activation in our well-characterized cohort of 117 T-ALL patient samples and related it to clinical and biological parameters including outcome. Results The NOTCH pathway model was calibrated using a microarray dataset containing high-grade serous ovarian cancer-which has high NOTCH activity-and normal ovarian tissue samples that lack NOTCH activity. Validation of the test using datasets from primary cells and cell lines of various origins revealed that it measures the NOTCH activity status in different cellular contexts. In primary diagnostic T-ALL samples, we observed a significant relationship between NOTCH pathway activity scores and active, intracellular cleaved NOTCH1 (ICN1) protein levels and the presence of NOTCH1-activating mutations. We next scored NOTCH pathway activity over the four T-ALL subgroups ETP-ALL, TLX, Proliferative and TALLMO. The TLX subgroup had the highest NOTCH activity levels compared to the other subgroups, consistent with the high percentage of TLX cases with NOTCH1/FBXW7 mutations. Strikingly, the significance of the correlation between ICN1 levels and NOTCH pathway activity was mainly attributed to the strong NOTCH1-activating mutations that include NOTCH1 juxtamembrane domain mutations, or hetero-dimerization mutations combined with PEST domain or FBXW7 mutations. When assessing the event-free survival and relapse-free survival curves, we observed that patients with the lowest (lower than the 25th-percentile) NOTCH pathway activity scores had the shortest event-free survival compared to the others (p<0.05, log-rank test). Summary/Conclusion High NOTCH pathway activation was mostly present in-but not limited to-T-ALL samples harboring strong NOTCH1 mutations, indicating that additional mechanisms can activate NOTCH signaling. Combined with the observation that the measured NOTCH pathway activity relates to ICN1 protein levels, this indicates that the pathway activity score more accurately reflects NOTCH pathway activity than the predicted activity based on NOTCH1 mutations alone. Disclosures Holtzer: Philips Research: Employment. Verhaegh:Philips Research: Employment. van de Stolpe:The Netherlands: Employment; Eindhoven: Employment; Philips Research: Employment.

Abstract 5129: Actionable activating oncogenic HER2 transmembrane and juxtamembrane domain mutations

Abstract Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) including G660D, R678Q, E693K and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade. Citation Format: Kanika Bajaj. Actionable activating oncogenic HER2 transmembrane and juxtamembrane domain mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5129.

FLT3mutation Assay Laboratory Cross Validation: Results from the CALGB 10603/Ratify Trial in Patients with Newly Diagnosed FLT3-Mutated Acute Myeloid Leukemia (AML)

FLT3mutation Assay Laboratory Cross Validation: Results from the CALGB 10603/Ratify Trial in Patients with Newly Diagnosed FLT3-Mutated Acute Myeloid Leukemia (AML)

In the literature: December 2018

The current development of immune checkpoint modulatory treatments has shown durable responses in the treatment of multiple cancer types.1 However, predictive biomarkers beyond PD-1 ligand 1 (PD-L1) expression and high microsatellite instability (MSI-H) to stratify patients and identify those who could benefit of these therapies are needed. In this sense, a recent study published in Science by Cristescu et al 2 describes the potential usefulness of combining the tumour mutational burden (TMB) and the T cell-inflamed gene expression profile (GEP) to jointly predict clinical response to pembrolizumab. Both PD-L1 and the GEP represent a T cell-inflamed tumour microenvironment (TME), whereas TMB and MSI-H are indirect measures of tumour antigenicity generated by somatic tumour mutations. In the mentioned study, TMB was evaluated by whole-exome sequencing (WES) of germline and tumour DNA, and the T cell-inflamed GEP was analysed by targeted gene expression profiling of tumour RNA (with the NanoString platform) from formalin fixed, paraffin-embedded pretreatment samples. To assess the individual and joint clinical utility of TMB and GEP, patients were stratified in four biomarker-defined clinical response groups based on predefined cut-offs for TMB and GEP. Objective response rates were strongest in patients with GEPhi TMBhi (37%–57%), moderate in those with GEPhi TMBlo (12% to 35%) and GEPlo TMBhi (11%–42%) and reduced or absent in those with GEPlo TMBlo (0%–9%). Additionally, longer PFS were seen in patients with higher levels of both TMB and GEP. Findings were comparable when TMB and PD-L1 expression were jointly assessed. Interestingly, more than 300 patient samples with advanced solid tumours and melanoma across 22 tumour types from four KEYNOTE clinical trials were included and analysed. Samples were assessed separately in four cohorts: one discovery, one pan-tumour validation and …

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

Targeting Oncogenic Signaling in Mutant FLT3 Acute Myeloid Leukemia: The Path to Least Resistance.

The identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly-targeted treatment strategies designed to improve outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3 is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD) present in 30–35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus, encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as a monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance occurs by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain (TKD) at residue Asp835 (D835) to form a ‘dual mutation’ (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades; however, little is known about which divergent signaling pathways are controlled by each of the FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways deregulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise of more durable and personalized therapeutic approaches to improve treatments of FLT3 mutant AML.

Abstract 806: KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation

Abstract More than 30% of patients with acute myeloid leukemia (AML) harbor a mutation with internal tandem duplication (ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain of FMS-like tyrosine kinase-3 (FLT3), which results in aberrant activation FLT3 by auto-phosphorylation, and confers poor prognosis. To develop novel FLT3 inhibitors, ten novel oxazole amine derivatives were designed and synthesized based on FLT3 protein structure. Among the synthesized small molecule compounds, KI2011 exhibited selectively inhibitory activities of the class III receptor tyrosine kinases, especially FLT3, wile-type, FLT3-D835Y mutant and FLT3-internal tandem duplication (ITD) mutant isoforms with IC50 values of 25, 9, and 1.2 nM, respectively, determined by an in vitro kinase activity assay. The compound decreased FLT3 phosphorylation and its downstream signaling in MV4-11 cells harboring FLT3-ITD mutation. Treatment of KI2011 selectively inhibited cell proliferation and induced apoptosis of MV411 cells in a concentration-dependent manner, but not in K562 (FLT3 negative) cells. In addition, we found that KI2011 enhanced radiosensitivity of MV4-11 cells. The results suggest that KI2011 is a novel potent FLT3 inhibitor that may be further developed in the preclinical studies as a therapeutic agent in AML treatments. Citation Format: Hwani Ryu, Jie-Young Song, Jiyeon Ahn. KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation [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 806.

FLT3 receptor/CD135 expression by flow cytometry in acute myeloid leukemia: Relation to FLT3 gene mutations and mRNA transcripts

BackgroundAlterations of the FLT3 gene are the most frequent molecular aberrations seen at diagnosis of acute myeloid leukemia (AML). Two main types of FLT3 mutations have been the most commonly detected; internal tandem duplication (ITD) in the juxtamembrane domain and point mutation D835Y in the tyrosine kinase domain (TKD). Both classes of mutations result in constitutive activation of FLT3 receptor/CD135. AimTo assess the frequency of FLT3 gene mutations (ITD and TKD D835Y) and the flow cytometric expression of FLT3 receptor/CD135 among AML patients to define the role for FLT3 receptor expression in predicting FLT3 gene mutational status and mRNA transcript level. Subjects and methodsEighty AML patients at diagnosis and 20 control subjects were enrolled. FLT3 receptor/CD135 expression, FLT3 gene mutations, and FLT3 transcript level were evaluated by flow cytometry, conventional polymerase chain reaction (PCR), and quantitative real-time reverse-transcription PCR, respectively. Fluorescence in situ hybridization was done to stratify patients into favorable, intermediate, and poor cytogenetic risk groups. ResultsFLT3-ITD was detected in 22.5% AML patients, while none had FLT3-TKD D835Y mutation. A cut-off value of >17% was assigned to define FLT3 receptor/CD135+ cases. FLT3 receptor/CD135 and FLT3 transcripts were overexpressed in 100% AML patients; higher levels were found among AML-M5 subtype and poor cytogenetic group. AML patients harboring FLT3-ITD showed a trend for higher FLT3 receptor/CD135 expression and FLT3 transcript level than those with wild-type FLT3. FLT3 receptor/CD135 >49% was predictive for FLT3-ITD. A positive correlation was found between FLT3 receptor/CD135 expression and FLT3 transcript level. ConclusionEvaluation of FLT3 receptor/CD135 expression by flow cytometry at diagnosis of AML could constitute a predictor for the FLT3-ITD mutational status and FLT3 transcript level.

The impact of FLT3 mutations on treatment response and survival in Chinese de novo AML patients

ABSTRACTObjective: Two distinct forms of FMS-like tyrosine kinase 3 (FLT3) mutations, internal tandem duplication (ITD) in the juxtamembrane domain and point mutation within the activation loop of the tyrosine kinase domain (TKD), have been identified in considerable number of patients with AML. This study was aimed to analyze the impacts of these mutations on clinical outcomes, and assess the efficacy of different therapeutic regimens (allo-HSCT, sorafenib, or conventional chemotherapy) for AML patients with FLT3 mutations after the standard induction therapy.Materials and methods: We analyzed DNA samples from 158 consecutive de novo AML patients (18–60 years, excluding APL) with FLT3 mutations between July 2010 and October 2015.Results: We found that AML patients with FLT3-TKD mutations have more favorable clinical outcomes than those with FLT3-ITD mutations. We also found that allo-HSCT therapy subgroup achieved longer OS and RFS than non-allo-HSCT therapy subgroup for FLT3-ITD positive patients (p < 0.001, p = 0.071). However, compared with the clinical outcomes in non-primary refractory patients, sorafenib did not show an obvious beneficial effect for the primary refractory patients. Further study on a large scale is still recommended.Conclusions: FLT3-TKD-mutated AML patients have more favorable clinical outcomes than those with FLT3-ITD mutations. Allo-HSCT therapy subgroup achieved longer OS and RFS than non-allo-HSCT therapy subgroup for FLT3-ITD positive patients. Compared with the clinical outcomes in non-primary refractory patients, sorafenib did not show an obvious beneficial effect for the primary refractory patients.

FLT3 Mutations in Pediatric Acute Promyelocytic Leukemia; A Report from the Children's Oncology Group AAML0631 Trial

BackgroundSurvival for acute promyelocytic leukemia (APL) has improved dramatically in recent decades through use of targeted therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Early deaths, principally due to complications of APL coagulopathy, remain a significant barrier to cure. White blood cell (WBC) count at diagnosis is a well validated risk marker for early death and relapse. While APL is characterized by t(15;17), mutations of the FLT3 gene are prevalent. Prior reports in both adult and pediatric APL have demonstrated variable impact of FLT3 mutation status on outcomes. In a pediatric cohort from the Intergroup Study CALGB 9710, our group previously found an association of FLT3 mutations with early death. The more recent Children's Oncology Group (COG) APL trial AAML0631 included addition of two courses of arsenic trioxide (ATO) during consolidation. Relapse rate on this study was low, but survival, particularly for the high risk patients, was impacted by early deaths. In a prior analysis we reported that a significant proportion of patients on AAML0631 experienced clinically significant bleeding and clotting events. Here we report our analysis of the prevalence of FLT3 mutations and their association with outcomes for pediatric APL patients treated on the AAML0631 study.MethodsDiagnostic bone marrow samples were collected from consenting patients with newly diagnosed APL enrolling onto COG AAML0631. FLT3 mutation testing of these bone marrow samples included evaluation for internal tandem duplications (FLT3/ITD) of the juxta-membrane domain and point mutations of the activating loop (FLT3/ALM) of the tyrosine kinase domain. FLT3 mutation status was correlated with clinically significant bleeding/clotting, early death during induction and relapse. Significance of correlation was determined using the chi-square test and relapse risk for patients who achieved CR by end of Induction 1 was calculated using cumulative incidence.Eligible patients were age ≥ 2 years and < 22 years and required confirmation of PML-RARA by PCR testing. Risk group was defined by WBC at diagnosis: standard risk (SR) WBC < 10,000 and high risk (HR) WBC ≥ 10,000. Treatment regimen has been previously reported [Kutny et al, ASH Abstract, 2015].ResultsThere were 101 evaluable patients on AAML0631 including 35 HR APL and 66 SR APL. A total of 94 patients had specimens available for FLT3 mutation testing. Patients with unknown FLT3 status (3 HR and 4 SR) were excluded from the following analyses. FLT3 mutations were present in 43% (N=40) including 35% (N=33) with FLT3/ITD, 4% (N=4) with FLT3/ALM, and 3% (N=3) with both FLT3/ITD and FLT3/ALM. The median WBC was 24,450 for FLT3 mutant APL compared to 7,130 for FLT3 wild type, and the rate of FLT3 mutation was significantly higher in HR APL compared to SR APL (66% vs. 31%, P=0.001). Four patients died during induction (all HR APL with WBC of 16200-173800), but early death did not correlate with FLT3 mutation (2/4 patients had FLT3 mutations). We analyzed association of FLT3 mutations with prevalence of clinically significant (Grade III-IV) bleeding or clotting events during induction. There was no difference in prevalence of coagulopathy events (N= 19) between those with and without FLT3 mutations (18% vs. 22%, P=0.57).While OS and EFS were not significantly different, the relapse rate from end of Consolidation 1 was significantly higher at 6% (N=3) in FLT3 mutant patients versus 0% in FLT3 wild-type (P=0.039) [Figure 1].ConclusionsFLT3 mutations are highly prevalent (43%) in pediatric APL. The COG AAML0631 study represents the second largest group of pediatric APL patients with outcome data treated on a cooperative group trial and to our knowledge the current report is the largest group of pediatric APL patients evaluated for outcome based on FLT3 mutation status. This study failed to show an association of FLT3 mutations with early death or bleeding/clotting events in induction. With ATO consolidation, the relapse rate was low on this trial. However, it is of great interest that the relapse rate following ATO consolidation was significantly higher in FLT3 mutant patients. Evaluation of a larger (international) cohort of pediatric APL patients is warranted to further define of role of FLT3 in relapse risk, but the current results give further support to consideration of integrating FLT3 inhibitors into relapsed/refractory APL treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Discovery and Functional Validation of Novel Pediatric Specific FLT3 Activating Mutations in Acute Myeloid Leukemia: Results from the COG/NCI Target Initiative

Mutations in the FLT3 gene are among the most common somatic events in AML, with a higher prevalence in adults than in children. The most common activating mutations of FLT3 include internal tandem duplications (FLT3/ITD) in the juxtamembrane domain (JMD) or missense mutations in the tyrosine kinase domain (TKD) at the D835/I836 positions (FLT3/ALM). To date, much of the data on FLT3 mutations has been derived from adult studies and comprehensive sequencing of the FLT3 gene from recent TCGA analysis demonstrated that FLT3 activating mutations were limited to the FLT3/ITD in the JMD and D835/I836 hotspots. As part of the Children's Oncology Group (COG)/NCI TARGET AML initiative, we interrogated the genomic landscape of pediatric AML and identified and verified novel FLT3 activating events that appear to be unique to childhood AML and could provide a target for therapeutic intervention.Whole genome sequencing was performed in a discovery cohort (N=200) followed by validation with targeted exome capture for a total of 799 diagnostic specimens from children treated on contemporary COG trials. In addition to the known FLT3 mutations (FLT3/ITD, N=128 and D835/I836, N=37), we identified novel point mutations (PM; N=49) and novel insertion-deletions (in-dels; N=12). We observed a prevalence of 7.6% of novel PMs and in-dels, in addition to the FLT3/ITD and D835/I836 mutations. The total prevalence of all FLT3 mutations was 28%. In contrast to adult AML, where virtually all non-ITD activating mutations are limited to the D835/I836 region, FLT3PMs in the pediatric cohort occurred in TKD domain (N=44), but commonly occurred in the transmembrane domain (TMD) and JMD. Twelve somatic mutations were identified at distinct positions within the JMD, the region involved in regulation of activity of the kinase. Among the JMD mutations, 9 occur at novel pediatric specific sites with significant activating potential (E573D/G, L576R, T582N, D586Y, Y589H, E596K/G, E598D, Y599C, D600G).Crystal structure analysis of FLT3 variants was used to assess the potential functional significance of the newly discovered variants. This structural modeling indicated that many of the mutations within the TMD (e.g. A680V) and TKD1 (L616R, M664I, M665L) were predicted to cause JMD destabilization, resulting in dysregulated activation of FLT3. Additionally, almost all JMD mutations have the potential to significantly disrupt the auto-inhibitory conformation, resulting in constitutive FLT3 activation. Mutations causing excessive activation of the kinase may have significant oncogenic capacity. In order to assess functional implications of the mutations, we cloned and expressed 6 of the most common novel variants (E573D, L576R, Y599C, D600G, F612L, and A680V) for functional assessment. Of the 6 mutations tested, 5 (E573D, L576R, Y599C, D600G, and A680V) resulted in auto-phosphorylation of FLT3, demonstrating dysregulated and enhanced kinase activation.Acquired mutations following tyrosine kinase inhibitor (TKI) exposure are heavily implicated in resistance and are almost exclusively confined to the two TKD regions, and the D835 position is a hotspot for resistance conferring mutations. It is important to identify patients at diagnosis who harbor dual mutations as this could indicate de novo resistance to TKIs, and exposure to these agents would have no efficacy, but may result in unnecessary toxicity. The presence of dual FLT3/ITD and FLT3/ALM mutations at diagnosis has been reported to occur at a very low prevalence. We analyzed the presence of co-occurring FLT3/ITDand FLT3 PMs in our pediatric cohort. Of the 128 patients with FLT3/ITD, 18 (14%) harbored a secondary FLT3 PM. Mutations in the JMD (N=8) and TMD (N=2) accounted for 56% of co-occurring mutations. Only 22% (N=4) mutations occurred at the D835/I836 sites, with an additional 22% (N=4) located at other TKD sites. Identified TKD diagnostic mutations included F612L, F616R, N676K/S, and N841K, a few which have only been reported in the setting of post TKI relapse.We identified novel pediatric specific FLT3 mutations with significant functional capacity. Importantly, these activating mutations may be uniquely susceptible to FLT3 inhibition and provide a therapeutic target in pediatric AML. Further work is ongoing to completely understand the oncogenic potential of each unique mutation and their prognostic and therapeutic implications. DisclosuresNo relevant conflicts of interest to declare.