Gilteritinib Treatment Research Articles

Gilteritinib Reduces FLT3 Expression in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a genetically diverse and challenging malignancy, with mutations in the FLT3 gene being particularly common and deleterious. Gilteritinib, a potent FLT3 inhibitor, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsed/refractory AML with FLT3 mutations. Although gilteritinib was developed based on its inhibitory activity against FLT3 kinase, it is important to understand the precise mechanisms of its antileukemic activity in managing drug resistance and discovering biomarkers. This study was designed to elucidate the effect of gilteritinib on the FLT3 expression level. The results showed that gilteritinib induced a dose-dependent decrease in both FLT3 phosphorylation and expression. This reduction was particularly pronounced after 48 h of treatment. The decrease in FLT3 expression was found to be independent of changes in FLT3 mRNA transcription, suggesting post-transcriptional regulatory mechanisms. Further studies were performed in various AML cell lines and cells with both FLT3 wild-type and FLT3 mutant exhibited FLT3 reduction by gilteritinib treatment. In addition, other FLT3 inhibitors were evaluated for their ability to reduce FLT3 expression. Other FLT3 inhibitors, midostaurin, crenolanib, and quizartinib, also reduced FLT3 expression, consistent with the effect of gilteritinib. These findings hold great promise for optimizing gilteritinib treatment in AML patients. However, it is important to recognize that further research is warranted to gain a full understanding of these mechanisms and their clinical implications in the context of FLT3 reduction.

Abstract 3115: Systematic analysis of gene mutations and therapeutic efficacy via ex vivo hematological vitroscreen platform

Abstract Acute myeloid leukemia (AML) is a common hematological malignancy found in adult patients, and FMS-like tyrosine kinase 3 gene (FLT3) mutation occurs in about 30% of patients. In 2019, a phase 3 clinical trial suggested that gilteritinib treatment resulted in significantly longer survival than salvage chemotherapy in relapsed or refractory FLT3-mutated AML patients. Targeting FLT3-mutants in AML has become one of the important therapeutic strategies in this decade. The ex vivo hematological VitroScreen platform provides a powerful tool to evaluate the performance of FLT3 inhibitors and other test articles. With Champions Oncology’s primary hematological repository (https://www.championsoncology.com/hematological-vitroscreen) and proprietary LUMIN Bioinformatics platform, we classified models based on patient responses, clinical annotation, NGS data, and cell surface markers. In this Ex vivo study, we tested gilteritinib in 30 AML patients, and 8 of models were identified as FLT-3 mutants’ carrier. Fold changes of cell viability after gilteritinib treatment were assessed based on quantitation of the ATP present. FLT3-mutants models showed significantly higher sensitivity to gilteritinib than FLT3-WT models (IC50, 132.56 nM vs. 234.16nM) in a 6-day assay. Only 25% of models in the FLT3-mutants group had higher IC50 than the average of all AML models (n=28, IC50 = 204.05 nM), compared to 53% in the FLT3-WT group. Our preclinical data highly match the observations in previous clinical trials, which suggest that VitroScreen is a reliable high throughput tool for drug development of drug resistant cancers. Citation Format: Fu-Ju Chou, Stefano Cairo, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. Systematic analysis of gene mutations and therapeutic efficacy via ex vivo hematological vitroscreen platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3115.

Gilteritinib as Post-Transplant Maintenance for AML With Internal Tandem Duplication Mutation of FLT3.

Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of FLT3 (FLT3-ITD) AML. These patients are routinely treated with a FLT3 inhibitor after HCT, but there is limited evidence to support this. Accordingly, we conducted a randomized trial of post-HCT maintenance with the FLT3 inhibitor gilteritinib (ClinicalTrials.gov identifier: NCT02997202) to determine if all such patients benefit or if detection of measurable residual disease (MRD) could identify those who might benefit. Adults with FLT3-ITD AML in first remission underwent HCT and were randomly assigned to placebo or 120 mg once daily gilteritinib for 24 months after HCT. The primary end point was relapse-free survival (RFS). Secondary end points included overall survival (OS) and the effect of MRD pre- and post-HCT on RFS and OS. Three hundred fifty-six participants were randomly assigned post-HCT to receive gilteritinib or placebo. Although RFS was higher in the gilteritinib arm, the difference was not statistically significant (hazard ratio [HR], 0.679 [95% CI, 0.459 to 1.005]; two-sided P = .0518). However, 50.5% of participants had MRD detectable pre- or post-HCT, and, in a prespecified subgroup analysis, gilteritinib was beneficial in this population (HR, 0.515 [95% CI, 0.316 to 0.838]; P = .0065). Those without detectable MRD showed no benefit (HR, 1.213 [95% CI, 0.616 to 2.387]; P = .575). Although the overall improvement in RFS was not statistically significant, RFS was higher for participants with detectable FLT3-ITD MRD pre- or post-HCT who received gilteritinib treatment. To our knowledge, these data are among the first to support the effectiveness of MRD-based post-HCT therapy.

Identification of Unexpected Responders to Gilteritinib in a Cohort of FLT3-WT AML Patients Using Ex-Vivo Testing

Introduction: Despite the numerous significant advances in leveraging actionable genetic biomarkers, targeted therapies continue to exhibit limited success in AML. This may be attributed to the genetic heterogeneity and plasticity of cancer cells, which ultimately overrides the driving force of individual mutations and their resulting phenotypes. In the face of this well-known challenge, there is a growing promise of functional assays that can leverage the integrated phenotypic response of cancer patients' cells to determine treatment schemes in a personalized manner. In addition, functional testing holds the potential to identify elusive responders to targeted therapies in cohorts of patients who do not present the actionable mutations that are used as biomarkers for prescription. Aim: To perform pre-clinical correlation studies between FLT3 mutational status and the ex-vivo response to Gilteritinib using a functional assay of Patient Micro Avatars (PMAs) developed at OncoPrecision. Materials and Methods: Ex-vivo testing with PMAs was performed to evaluate the ex-vivo response to Gilteritinib in 67 bone marrow or peripheral blood samples from patients diagnosed with different AML subtypes (de novo, relapsed/refractory and secondary). Pathological subpopulations were analyzed using lineage markers and survival of blasts was assessed using flow cytometry. Samples were classified as “responders” and “non-responders” using in-house supervised and non-supervised machine learning tools. In parallel, samples were analyzed by NGS and molecular biology to identify mutations and internal tandem duplications in the FLT3 gene. Finally, FLT3 alterations were correlated with the phenotypic response obtained in the functional assay using statistical analysis to compare mutant and non-mutant frequencies in the two groups. Results: The results of the phenotypic screening revealed that ~70% of patients with FLT3 mutations exhibited a strong response to Gilteritinib, which is in line with the presence of the actionable genetic biomarker (Figure). The fact that the remaining ~30% of mutated patients presented a poor response to Gilteritinib suggests the existence of acquired mechanisms of resistance that are dominant over the mutational status of FLT3. Interestingly, we also identified a large group of FLT3wt patients (~30%) which showed a phenotypic response to Gilteritinib equivalent to the one observed in FLT3mut patients (Figure). This finding unveils the existence of a subpopulation of FLT3wt patients who may benefit from Gilteritinib treatment and cannot be identified using conventional genetic testing. Conclusions: The present study highlights the reach of functional testing as a powerful tool to predict Gilteritinib resistance in FLT3mut backgrounds and to identify exceptional responders in FLT3wt backgrounds, creating opportunities for AML patients beyond the currently available genetic biomarkers.

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.

STI-8591, a Novel Tyrosine Kinase Inhibitor, Shows Excellent Antileukemic Activity in FLT3-Mutated (mut) Acute Myeloid Leukemia (AML)

FMS-related tyrosine kinase 3 ( FLT3) mutations, including internal tandem duplications ( FLT3-ITD) and tyrosine kinase domain ( FLT3-TKD), are found in approximately 30% of AML patients. FLT3 has emerged as a promising molecular target in therapy of AML. Herein, we report on the activity of STI-8591, a novel kinase inhibitor developed by Hangzhou ACEA Pharmaceutical Research Co, in AML models carrying FLT3 mut, including those associated with TKI resistance [e.g., FLT3-ITD(F691L)]. We first determined the in vitro antileukemic effects of STI-8591 in FLT3mut human AML cell lines (MV4-11, MOLM-13), a murine cell line Ba/F3 transduced with FLT3-TKD(D835Y)[Ba/F3 FLT3-TKD(D835Y)] or FLT3-ITD-TKD(D835Y) [Ba/F3 FLT3-ITD-TKD(D835Y)] or FLT3-ITD/F691L [Ba/F3 FLT3-ITD(F691L), which has been associated with drug-resistant, and primary blasts from FLT3mut AML patients. STI-8591 effectively inhibited cell proliferation and induced apoptosis in FLT3-ITD/TKDmut cell lines (IC 50: 0.33 to 9.19nM at 24 hours). Of note, STI-8591 demonstrates 4-7-fold higher antileukemic activity on otherwise resistant Ba/F3 FLT3-ITD(F691L) cells than the currently available FLT3 inhibitors, Gilteritinib and Quizartinib. Similar antileukemic activity of STI-8591 was observed on primary FLT3mut AML blasts (median IC 50: 13.67nM, n=5). The antileukemic mechanism of STI-8591 is associated with inhibition of the phosphorylation of the mutated FLT3 protein and its downstream targets (i.e., STAT5, AKT, and ERK) as shown by western blot analysis. To test the in vivo antileukemic activity of STI-8591, we utilized 4 FLT3mut AML murine and human models, obtained by transplanting: 1. BM mononuclear cells (MNCs) from CD45.2 MLLPTD/WTFLT3ITDITD AML mice congenic CD45.1 B6 mice; 2. murine cell lines [i.e. Ba/F3 FLT3-TKD(D835Y) , Ba/F3 FLT3-ITD-TKD(D835Y), or Ba/F3 FLT3-ITD(F691L), 3. Luciferase-expressing FLT3mut MOLM13 cells (MOLM13 Luci) into NCG mice; 4. primary blasts from FLT3 mut AML patients into NSGS mice. We treated these mice for 3-4 weeks [3 to 30mg/kg/day(d), oral gavage]. For the MLLPTD/WTFLT3ITDITD model (n=10 mice/group), compared with Vehicle(V)-treated mice [median survival: 44 d], we observed significantly lower engraftment and longer overall survival (OS) in mice treated with STI-8591 10mg/kg (52 d, p<0.0001) or 30mg/kg (57 d, p<0.0001) or Gilteritinib 10mg/kg (49 d, p=0.0029). At 10mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0195). Treatment of STI-8591 also demonstrated significant benefits to mice transplanted with FLT3mut Ba/F3 cells. For the TKI-resistant Ba/F3 FLT3-ITD(F691L) model (n=10/group), STI-8591 (median:19 and 37 d for 10 and 30 mg/kg) and Gilteritinib (17 and 24 d for 10 and 30 mg/kg) -treated mice had longer OS than V-treated (12 d) mice. At equivalent dosage (10 and 30mg/kg), STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0013 and p<0.0001). For the MOLM13 Luci+ model (n=5/group), mice receiving STI-8591 or Gilteritinib treatment demonstrated a significant reduction in disease burden as observed by bioluminescence imaging (BLI), and prolonged OS. Compared with V-treated mice (median: 23 d), we observed longer OS in Gilteritinib (10mg/kg, 33 d, p=0.0023) and STI-8591 (3mg/kg, 27 d, p=0.0179; 10mg/kg, 34 d, p=0.0023; and 30mg/kg, 35 d, p=0.0023) -treated mice. At 10 mg/kg dosage, STI-8591- and Gilteritinib- treated mice showed no significant difference in OS (p=0.2411). Similar experiments are ongoing in FLT3 mut primary AML patient-derived xenografts (PDXs) and will be reported at the ASH meeting. To evaluate antileukemic activity of STI-8591 in mice bearing disease in central nervous system (CNS), we implanted 5×10 5 /3 µL MOLM13 cells intracranially into SCID mice to construct the CNS leukemia model (n=9/group). Compared with V-treated mice (median: 11 d), we observed a longer OS in mice treated with Gilteritinib (10 mg/kg, 18 d, p=0.0004) or STI-8591 (10 mg/kg, 23 d, p<0.0001; and 30 mg/kg, 26 d, p<0.0001). At 10 mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.005). In conclusion, STI-8591, a novel TKI exhibited a potent antileukemic activity in various FLT3mut AML models, including those carrying FLT3 mut associated with drug resistance. These results supported further clinical development of STI-8591 for treatment of AML.

Gilteritinib and the risk of intracranial hemorrhage: a case series of a possible, under-reported side effect

Gilteritinib is currently approved for patients with relapsed/refractory AML with FLT3 mutations, based on the positive results of the pivotal ADMIRAL study. In ADMIRAL trial, no increased risk of bleeding was reported, but in the previous dose finding study, a single event of intracranial hemorrhage (ICH) was registered after exposure to subtherapeutic doses of gilteritinib. Here, we report the first case series on five ICHs diagnosed in patients with FLT3-mutated AML, occurred within the first month of exposure to gilteritinib. Our cohort included 24 patients treated in three Italian centers. Most of these ICH cases were non-severe and self-limiting, while one was fatal. This link with ICHs remains in any case uncertain for the presence of active AML. We further reported that an analysis of the post-marketing surveillance data (EudraVigilance) retrieved other 11 cases of ICHs present in the database after gilteritinib treatment. A causality assessment was performed according to the Dx3 method to evaluate the possibility that ICHs might be an actual side effect of gilteritinib. In conclusion, further research is needed to elucidate the potential role of gilteritinib in the pathogenesis of ICHs.

Prognostic value of the mutation types and dynamics of FLT3-ITD in acute myeloid leukemia.

The prognostic value of the mutation types and dynamics of FLT3-ITD in acute myeloid leukemia (AML) and other known factors were studied. Initial and follow-up samples from 45 AML patients with FLT3-ITD mutations were analyzed by fragment length analysis, Sanger sequencing, and next-generation sequencing. Some patients (13%) had multiple FLT3-ITD mutations, and many of them had acute promyelocytic leukemia (APL). FLT3-ITD mutations were classified according to mutation types, including duplication-only FLT3-ITD (52%) and FLT3-ITD with duplications and insertions (dup + ins) (48%). The dup + ins FLT3-ITD variant was independently associated with poor prognosis among non-APL patients (odds ratio, 2.92) in addition to FLT3-ITD with ≥50% variant allele frequency (VAF). The VAFs of FLT3-ITD were low (median 2.2%) when detected during morphologic complete remission (CR) after conventional chemotherapy; however, in two patients treated with gilteritinib after relapse, the VAFs of FLT3-ITD were much higher (>95% and 8.1%) in the morphologic CR state. The type of FLT3-ITD mutation is important in prognosis, and the dup + ins type of FLT3-ITD can be an indicator of poor prognosis. In addition, the FLT3-ITD mutation status may unexpectedly not match the morphologic examination results after gilteritinib treatment.

Severe Gastrointestinal Toxicity Following the Use of Gilteritinib: A Case Series and Analysis of Postmarketing Surveillance Data.

Gilteritinib has been approved as monotherapy in adults with acute myeloid leukemia (AML) FLT3 mutated with relapsed or refractory disease, in light of its advantages in terms of survival and the favorable safety profile. Hepatobiliary disorders and musculoskeletal and connective tissue disorders represent the most frequent adverse reactions associated with gilteritinib, whereas the most frequent serious adverse reaction is acute kidney injury. In the summary of product characteristics, gastrointestinal (GI) events are indicated as very common, in particular diarrhea, nausea and stypsis. Furthermore, serious GI disorders have been observed with gilteritinib in clinical trials, including GI hemorrhage, GI perforation and GI obstruction. However, the association with the FLT3 inhibitor has not been confirmed. Nevertheless, serious GI AEs have been recognized as an important potential risk to be monitored in postmarketing surveillance. We present three cases of serious self-limiting GI events observed in patients on gilteritinib treatment for AML, and an analysis of relevant available postmarketing surveillance data.

Abstract B019: ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML

Abstract The internal tandem duplication of the FLT3 receptor (FLT3-ITD) leads to constitutive receptor activation and is one of the most common mutations (~30%) in acute myeloid leukemia (AML), resulting in poor prognosis. Multiple FLT3 inhibitors (FLT3i) have been developed, including the potent FDA-approved FLT3i, gilteritinib. However, AML patients only respond to gilteritinib for approximately 6 months due to the emergence of drug resistance. Through an approach that integrated genomic, metabolomic, proteomic, and phosphoproteomic analyses with complementary genetic and pharmacologic screens, we demonstrated that gilteritinib resistance occurs in a stepwise manner with two distinct phases of early and late resistance (Joshi et al., Cancer Cell, 2021). While gilteritinib efficiently eliminates leukemia cells in the blood, residual cells survive within the bone marrow, where they are protected by extrinsic microenvironmental factors fostering early resistance. These lingering cells eventually develop intrinsic mutations in NRAS that allow them to resist gilteritinib treatment, leading to late resistance. Acquisition of mutations in the RAS pathway, particularly NRAS, are a common mode of late resistance (~11%) for many patients that relapse on FLT3i. Developing therapeutics to block NRAS activity remains a challenge with the lack of direct small-molecule inhibitors. Indirect approaches to inhibit mutant NRAS/MAPK pathway signaling have also not met much clinical success. Hence, therapeutic nucleic acid-based approaches, including antisense oligonucleotides (ASOs), may offer an alternative approach to target NRAS-driven cancers. We evaluated the effectiveness of a novel ASO targeting NRAS mRNA to restore gilteritinib sensitivity in resistant cultures in vitro and in primary AML patient samples ex vivo. We first assessed the potency of the NRAS ASO in both gilteritinib-sensitive and late resistant MOLM-14 AML cell lines (N=9) that expressed wild-type or mutant NRAS (G12S or G12D) via free uptake of ASO. NRAS expression was evaluated by qPCR after 48 hours. ASO treatment down-regulated mutant NRAS mRNA expression in a dose-dependent manner with an IC50 of < 1 µM and decreased NRAS protein levels. No changes in NRAS mRNA or protein levels were observed with non-targeting ASO control. Decreased NRAS mRNA expression restored sensitivity to gilteritinib in late-resistant cultures, leading to decreased cell viability and increased apoptosis. A similar trend was observed in primary AML cells from patients (N=2) that relapsed on gilteritinib, where reduction of approximately 50% of mutant NRAS mRNA robustly sensitized patient cells to gilteritinib. In contrast, the combination of gilteritinib and the MEK inhibitor trametinib was not as effective in either cell cultures or patient samples. Our results suggest that ASO-mediated knockdown of NRAS may circumvent the development of resistance and improve the durability of FLT3i. Citation Format: Sunil K. Joshi, Janét Pittsenbarger, Xiaolin Luo, Hadi Maazi, Alexey Revenko, Brian J. Druker, Elie Traer. ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B019.

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.

Gilteritinib Activity in Refractory or Relapsed FLT3-Mutated Acute Myeloid Leukemia Patients Previously Treated By Intensive Chemotherapy and Midostaurin: A Study from the French AML Intergroup ALFA/Filo

Background The recent phase 3 ADMIRAL trial has shown that outcomes of relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) patients were improved by the single-agent FLT3 tyrosine kinase inhibitor gilteritinib as compared with salvage chemotherapy. In this trial, overall survival (OS) was significantly improved in the gilteritinib arm and complete remission (CR/CRi), after excluding those obtained after allogenic hematopoietic stem cell transplantation (Allo-HSCT) were 26.3% in the gilteritinib group. Methods Here, the real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients (Cohort A) from the French early access program ELEGANCE between March 1st, 2019, and March 1st, 2021. Gilteritinib was administered to 27 patients in association with other treatments, while 140 received it as single agent (Cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (Cohort C). Results Characteristics of the three cohorts at gilteritinib initiation are described in Table 1. Briefly, in the whole cohort A, there were 141 (84.4%) cases of de novo AML, FLT3 mutation was ITD in 104 (62.3%) and TKD in 33 (19.8%), whereas 36 (21.6%) patients were FLT3 wild-type at diagnosis, acquired mutation later in disease evolution and were FLT3 mutated at gilteritinib initiation. Finally, 6 (3.6%) patients had both FLT3-ITD and TKD mutations. The median FLT3-ITD/wt ratio was 0.50 (IQR, 0.12-0.70 and range 0.01-1.90), and the median size of ITD was 48 bp (IQR, 30-75, range 3-408). Most patients (136, 81.4%) had an intermediate cytogenetic risk, and 84 (50.3%) had an NPM1 co-mutation. The main differences in patient characteristics in this study, compared to ADMIRAL, were ECOG ≥ 2 (83.6% vs 16.6%), FLT3-TKD mutations (21.0% vs 8.5%), primary induction failure (15.0% vs 40.0%) and previous lines of treatment (beyond 2nd in 37.1% vs 0.0%). The rates of response to gilteritinib are described in Table 1. Moreover, CRc (CR + CR without hematological recovery + CR with incomplete platelet recovery) to gilteritinib as single agent in subgroups defined as per AML phase at gilteritinib initiation were 4.5% (N=22) for patients refractory after front-line, 37.5% (N=32) for those relapsing less than 6 months from CR/CRi, 26.7% (N=30) when relapse occurred after 6 months and 16.1% (N=56) for patients refractory after 1st relapse and beyond. Finally, 32 (19.2%), 25 (17.9%) and 15 (22.4%) patients received an Allo-HSCT after gilteritinib, in CRc for 21 (65.6%), 17 (68.0%) and 11 (73.3%) patients in cohorts A, B and C, respectively. In terms of safety, in the subgroup of patients treated with gilteritinib as single agent (Cohort B), 39 (28.3%) experienced a serious adverse event (SAE) during gilteritinib treatment: infection in 22 (56.4%), hemorrhage in 1 (2.6%), cardiovascular in 4 (10.3%), hepatic in 4 (10.3%), differentiation syndrome in 2 (5.2%) and other SAE in 6 (15.4%). In terms of health care resource consumption, the median duration of hospitalization during the first 6 months of treatment was 9.0 days (IQR 0-29, range 0-117) with a median of 7 RBC units (IQR 0-14, range 0-54) and of 5 platelet units (IQR 0-13.5, range 0-56) transfusions. After a median follow-up of 14.5 months, median OS and 12-month OS were 6.4 months (IQR, 3.2-14.7) and 31.4% (95%CI 22.4-39.7) in the 140 patients who received gilteritinib as single agent, and 7.8 months (IQR, 3.2-20.8) and 38.3% (95%CI 26.0-50.4) in the subgroup of 67 patients previously treated by intensive chemotherapy and midostaurin (Figure 1). Multivariate analyses disclosed female gender (HR 1.61, 95%CI 1.07-2.42, p=0.02), adverse cytogenetic risk (HR 2.52, 95%CI 1.24-5.13, p=0.01) and allo-HSCT after gilteritinib (HR 0.13, 95%CI 0.05-0.37, p<0.0001) as factors significantly and independently associated with OS. Conclusion Although patients in this study were more heavily pretreated, these real-world data reproduce ADMIRAL results and provide new insights in the outcome of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Implementing RNAi Therapy Targeting FLT3 in Acute Myeloid Leukemia (AML)

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled (Gebru et. al. J. Hematol. Oncol. 2020). RNA interference (RNAi), mediated by short interfering RNA (siRNA) presents a novel therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. The success of RNAi therapy to a great extent is dictated by the siRNA delivery vehicle employed. This study explored the use of a lipid-substituted low molecular weight polyethyleneimine (lipopolymer) for delivering FLT3 siRNA in FLT3-ITD positive AML models and assess the efficacy of combining this treatment with chemotherapeutic drugs currently used in clinic. Low molecular weight polyethyleneimine (PEI) polymers were modified with aliphatic lipids to yield lipopolymers that self-assembled into nanocomplexes with siRNA. Treatment of MV4-11 cells with lipopolymer/FLT3 siRNA nanocomplexes resulted in ~50% reduction in cell proliferation rates (p<0.001), as determined by taking direct cell counts after the siRNA treatment. Quantitative analysis of relative mRNA transcript levels conducted by qPCR revealed downregulation of FLT3 gene by 30% (p<0.01). Furthermore, a similar decline (~35%, p<0.005) in FLT3 protein levels was observed when measured by flow cytometry through immunostaining with an anti-FLT3 antibody. Evaluation of apoptosis by FITC-Annexin V/propidium iodide (PI) staining showed a 3-fold increase in percentage of cells in the late apoptosis stage after FLT3 specific siRNA treatment (p<0.005). The effect of combining lipopolymer/FLT3 siRNA treatment with daunorubucin manifested as a decline in cell proliferation rates as well as 3-fold increase in apoptosis (p<0.05). To assess the impact of combination treatment on stem cells, colony forming ability was measured by seeding treated cells in methylcellulose and the colonies generated subsequently were counted after two weeks. A notable drop in colony counts was observed in the combination relative to individual treatments. However, similar analyses with the FLT3 targeting TKI midostaurin led to only a modest augmentation of anti-leukemic activity. Subsequently, the more recently approved FLT3 targeting TKI gilteritinib was explored in combination with lipopolymer/FLT3 siRNA nanocomplexes both in vitro and in vivo. For in vivo studies, lipopolymer/FLT3 siRNA nanocomplexes were administered via tail-vein injection at a siRNA dose of 25 µg per mouse (~1 mg/kg) once the tumor volume was ≥50mm3 in AML MV4-11 subcutaneous xenograft models created in male NCG (ABL/VAF) mice. Four injections of the nanocomplexes were administered, each 72 hours apart with simultaneous measurements of the tumor volume. Gilteritinib was administered four times intraperitonially at a dose of 5 mg/kg, with a spacing of 72 hours between doses. A significant reduction in tumor volume was observed in the combination treated tumors compared to gilteritinib alone treated tumors. Refractory/relapsed tumors underwent a second round of treatment (with both nanocomplexes and gilteritinib being administered four times each). The combination therapy significantly increased median survival to 65 days as compared to 45 days with gilteritinib treatment alone (p<0.05). These findings demonstrate the promising potential of RNAi implemented with lipopolymer nanocomplexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML.

Targeting OXPHOS de novo purine synthesis as the nexus of FLT3 inhibitor-mediated synergistic antileukemic actions.

Using a genome-wide CRISPR screen, we identified CDK9, DHODH, and PRMT5 as synthetic lethal partners with gilteritinib treatment in fms-like tyrosine kinase 3 (FLT3)–internal tandem duplication (ITD) acute myeloid leukemia (AML) and genetically and pharmacologically validated their roles in gilteritinib sensitivity. The presence of FLT3-ITD is associated with an increase in anaerobic glycolysis, rendering leukemia cells highly sensitive to inhibition of glycolysis. Supportive of this, our data show the enrichment of single guide RNAs targeting 28 glycolysis-related genes upon gilteritinib treatment, suggesting that switching from glycolysis to oxidative phosphorylation (OXPHOS) may represent a metabolic adaption of AML in gilteritinib resistance. CDK9i/FLT3i, DHODHi/FLT3i, and PRMT5i/FLT3i pairs mechanistically converge on OXPHOS and purine biosynthesis blockade, implying that targeting the metabolic functions of these three genes and/or proteins may represent attractive strategies to sensitize AML to gilteritinib treatment. Our findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and a rationale for a clinical trial of these novel combinations.

Gilteritinib monotherapy for relapsed/refractory FLT3 mutated acute myeloid leukemia: a real-world, multi-center, matched analysis.

Patients with FLT3-mutated relapsed or refractory (R/R) acute myeloid leukemia (AML) have a dismal prognosis. Gilteritinib is a FLT3 tyrosine kinase inhibitor (TKI) recently approved for patients with R/R AML. We aimed to characterize real-world data regarding gilteritinib treatment in FLT3-mutated R/R AML and to compare outcomes with matched FLT3-mutated R/R AML patients treated with chemotherapy-based salvage regimens. Twenty-five patients from six academic centers were treated with gilteritinib for FLT3-mutated R/R AML. Eighty percent were treated with a prior intensive induction regimen and 40% of them received prior TKI therapy. Twelve patients (48%) achieved complete response (CR) with gilteritinib. The estimated median overall survival (OS) of the entire cohort was eight (CI 95% 0-16.2) months and was significantly higher in patients who achieved CR compared to those who did not (16.3months, CI 95% 0-36.2 vs. 2.6months, CI 95% 1.47-3.7; p value = 0.046). In a multivariate cox regression analysis, achievement of CR was the only predictor for longer OS (HR 0.33 95% CI 0.11-0.97, p = 0.044). Prior TKI exposure did not affect OS but was associated with better event-free survival (HR 0.15 95% CI 0.03-0.71, p = 0.016). An age and ELN-risk matched comparison between patients treated with gilteritinib and intensive salvage revealed similar response rates (50% in both groups); median OS was 9.6months (CI 95% 2.3-16.8) vs. 7months (CI 95% 5.1-8.9) in gilteritinib and matched controls, respectively (p = 0.869). In conclusion, in the real-world setting, gilteritinib is effective, including in heavily pre-treated, TKI exposed patients.

P578: GILTERITINIB FOR RELAPSED/REFRACTORY FLT3 MUTATED ACUTE MYELOID LEUKEMIA A REAL-WORLD, MULTI-CENTER, MATCHED ANALYSIS

Background: Patients with FLT3-mutated relapsed or refractory (R/R) acute myeloid leukemia (AML) have a dismal prognosis. Gilteritinib is a FLT3 tyrosine kinase inhibitor (TKI) recently approved for patients with R/R AML. Aims: We aimed to characterize real-world data regarding gilteritinib treatment in FLT3-mutated R/R AML and to compare outcomes with matched FLT3-mutated R/R AML patients treated with chemotherapy-based salvage regimens. Methods: Survival analysis was performed by Kaplan Meyer with log rank test to compare between subgroups. Cox proportional hazards regression models were fitted to predict effect of covariates on overall survival (OS) and event free survival (EFS). All statistical analyses used 2-sided p value with a threshold of 0.05. Results: Twenty-five patients from six academic centers were treated with gilteritinib for FLT3-mutated R/R AML. The median age was 58 (IQR1-3 47-73) years, 80% (n=20) were treated with a prior intensive induction regimen and 40% of them received prior TKI therapy. After a median time of seven months post gilteritinib initiation (range 1-34), 12 patients (48%) achieved complete response (CR) with gilteritinib (figure 1A). The estimated median overall survival (OS) of the entire cohort was eight (CI 95% 0-16.2) months and was significantly higher in patients who achieved CR compared to those who did not (16.3 months, CI 95% 0-36.2 vs. 2.6 months, CI 95% 1.47-3.7; p value=0.046, figure 1B). In a multivariate cox regression analysis, achievement of CR was the only predictor for longer OS (HR 0.33 95% CI 0.11-0.97, p=0.044). Prior TKI exposure did not affect OS but was associated with better event-free survival (HR 0.15 95% CI 0.03-0.71, p=0.016). An age and ELN-risk matched comparison between patients treated with gilteritinib and intensive salvage revealed similar response rates (50% in both groups); median OS was 9.6 months (CI 95% 2.3-16,8) vs. 7 months (CI 95% 5.1-8.9) in gilteritinib and matched controls, respectively (p = 0.869, figure 1C). Image:Summary/Conclusion: In the real-world setting gilteritinib is effective, including in heavily pre-treated, TKI exposed patients.

Gilteritinib Enhances Anti-Tumor Efficacy of CDK4/6 Inhibitor, Abemaciclib in Lung Cancer Cells.

Abemaciclib is a cyclin-dependent kinases 4/6 (CDK4/6) inhibitor approved for the treatment of metastatic breast cancer. Preclinical studies suggest that abemaciclib has the potential for lung cancer treatment. However, several clinical trials demonstrate that monotherapy with abemaciclib has no obvious superiority than erlotinib to treat lung cancer patients, limiting its therapeutic options for lung cancer treatment. Here, we show that the US Food and Drug Administration (FDA)-approved drug, gilteritinib, enhances the cytotoxicity of abemaciclib through inducing apoptosis and senescence in lung cancer cells. Interestingly, abemaciclib in combination with gilteritinib leads to excessive accumulation of vacuoles in lung cancer cells. Mechanistically, combined abemaciclib and gilteritinib induces complete inactivation of AKT and retinoblastoma (Rb) pathways in lung cancer cells. In addition, RNA-sequencing data demonstrate that combination of abemaciclib and gilteritinib treatment induces G2 phase cell-cycle arrest, inhibits DNA replication, and leads to reduction in homologous recombination associated gene expressions. Of note, abemaciclib-resistant lung cancer cells are more sensitive to gilteritinib treatment. In a mouse xenograft model, combined abemaciclib and gilteritinib is more effective than either drug alone in suppressing tumor growth and appears to be well tolerated. Together, our findings support the combination of abemaciclib with gilteritinib as an effective strategy for the treatment of lung cancer, suggesting further evaluation of their efficacy is needed in a clinical trial.

Interim results from a postmarketing surveillance study of patients with FLT3-mutated relapsed/refractory AML treated with the FLT3 inhibitor gilteritinib in Japan.

ObjectiveGilteritinib received approval for the treatment of FLT3-mutated relapsed or refractory acute myeloid leukemia in Japan in 2018. In accordance with regulatory requirements, we conducted a multicenter, observational surveillance of gilteritinib use in Japan.MethodsPatients were followed for 6 months from gilteritinib treatment initiation. The primary endpoint of the surveillance was incidence of adverse drug reactions related to each element of the safety specification defined in the Japanese Risk Management Plan. This interim analysis presents data collected from 3 December 2018 to 20 September 2020.ResultsAmong 204 patients with case report forms, 107 consented to data publication. Of these 107 patients, 59.8% (n = 64) were male and 58.9% (n = 63) were aged ≥65 years; most received a 120-mg/day initial (80.4%; 86/107) and maximum (74.8%; 80/107) daily dose. The discontinuation rate was 61.7% (66/107); the most common reasons for discontinuation were disease progression (18.7%), transplantation (16.8%) and adverse events (15.0%). The adverse drug reaction rate was 77.6%. The incidences of adverse drug reactions (grade ≥ 3) related to each element of the safety specification were myelosuppression, 44.9% (38.3%); liver function disorder, 24.3% (6.5%); infections, 24.3% (21.5%); prolonged QT interval, 10.3% (2.8%); hemorrhage, 9.3% (6.5%); renal dysfunction, 6.5% (0); hypersensitivity, 5.6% (1.9%); interstitial lung disease, 4.7% (3.7%); cardiac failure/pericarditis/pericardial effusion, 1.9% (0.9%); pancreatitis, 0.9% (0); posterior reversible encephalopathy syndrome, 0.9% (0.9%). The composite complete remission rate was 62.7%; the 6-month overall survival rate was 77.7%.ConclusionGilteritinib treatment for 6 months in Japan was associated with acceptable efficacy and no new safety concerns were observed.

Molecular profile of FLT3-mutated relapsed/refractory patients with AML in the phase 3 ADMIRAL study of gilteritinib

AbstractThe phase 3 Study of ASP2215 Versus Salvage Chemotherapy in Patients With Relapsed or Refractory Acute Myeloid Leukemia (AML) With FMS-like Tyrosine Kinase (FLT3) Mutation (ADMIRAL) trial demonstrated the superiority of the FLT3 inhibitor, gilteritinib, to salvage chemotherapy (SC) in patients with FLT3-mutated relapsed or refractory (R/R) AML. Baseline comutations, FLT3-internal tandem duplication (ITD) allelic ratio and length, and treatment-emergent mutations were analyzed in patients in the ADMIRAL trial. Baseline comutations were grouped according to gene subgroups (DNA methylation/hydroxymethylation, transcription, chromatin–spliceosome, receptor tyrosine kinase-Ras signaling, TP53-aneuploidy, NPM1, DNMT3A, DNMT3A/NPM1, WT-1, and IDH1/IDH2). Across all but 1 gene subgroup (TP53-aneuploidy), higher pretransplant response rates and a trend toward longer overall survival were observed with gilteritinib vs SC. Patients with DNMT3A/NPM1 comutations who received gilteritinib had the most favorable outcomes of any molecular subgroup analyzed. Survival outcomes with gilteritinib were not adversely affected by FLT3-ITD allelic ratio, FLT3-ITD length, or multiple FLT3-ITD mutations. Among patients who relapsed on gilteritinib, Ras/mitogen-activated protein kinase (MAPK) pathway and FLT3 F691L gene mutations were the most common mutational events associated with treatment resistance. However, the occurrence of Ras/MAPK pathway gene mutations at baseline did not preclude a clinical benefit from gilteritinib. Acquisition of multiple Ras/MAPK pathway gene mutations at relapse suggests a high level of pathway reactivation is needed to overcome the gilteritinib treatment effect. These findings provide insight into the R/R AML molecular profile and the impact of FLT3 inhibitors on mutational evolution associated with treatment resistance and benefit of gilteritinib across a wide spectrum of molecular and genetic subgroups in FLT3-mutated R/R AML. This trial was registered at www.clinicaltrials.gov as #NCT02421939.

High-Dimensional Analysis Identifies Mechanisms of Gilteritinib Resistance in FLT3-Mutated AML

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. DisclosuresBlachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.