Catherine C. Smith

Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia

Effective targeted cancer therapeutic development depends upon distinguishing disease-associated ‘driver’ mutations, which have causative roles in malignancy pathogenesis, from ‘passenger’ mutations, which are dispensable for cancer initiation and maintenance. Translational studies of clinically active targeted therapeutics can definitively discriminate driver from passenger lesions and provide valuable insights into human cancer biology. Activating internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in approximately 20% of acute myeloid leukaemia (AML) patients and are associated with a poor prognosis. Abundant scientific and clinical evidence, including the lack of convincing clinical activity of early FLT3 inhibitors, suggests that FLT3-ITD probably represents a passenger lesion. Here we report point mutations at three residues within the kinase domain of FLT3-ITD that confer substantial in vitro resistance to AC220 (quizartinib), an active investigational inhibitor of FLT3, KIT, PDGFRA, PDGFRB and RET; evolution of AC220-resistant substitutions at two of these amino acid positions was observed in eight of eight FLT3-ITD-positive AML patients with acquired resistance to AC220. Our findings demonstrate that FLT3-ITD can represent a driver lesion and valid therapeutic target in human AML. AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.

The total influenza vaccine failure of 1947 revisited: Major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic

Although vaccine-induced immunity to influenza A virus is continually challenged by progressively selected mutations in the viruss major antigens (antigenic drift), virus strains within a subtype (e.g., H1N1) are antigenically cross-reactive. Although cross-immunity diminishes as further mutations accumulate, necessitating frequent changes in vaccine strains, older vaccines are usually partially protective. The post-World War II epidemic of 1947 is notable for the total failure of a vaccine previously effective in the 1943–44 and 1944–45 seasons. We have combined extensive antigenic characterization of the hemagglutinin and neuraminidase antigens of the 1943 and 1947 viruses with analysis of their nucleotide and amino acid sequences and have found marked antigenic and amino acid differences in viruses of the two years. Furthermore, in a mouse model, vaccination with the 1943 vaccine had no effect on infection with the 1947 strain. These findings are important, because complete lack of cross-immunogenicity has been found previously only with antigenic shift, in which antigenically novel antigens have been captured by reassortment of human and animal strains, sometimes leading to pandemics. Although the 1947 epidemic lacked the usual hallmarks of pandemic disease, including an extensive increase in mortality, it warns of the possibility that extreme intrasubtypic antigenic variation (if coupled with an increase in disease severity) could produce pandemic disease without the introduction of animal virus antigens.

Crenolanib is a selective type I pan-FLT3 inhibitor

Significance Rapid evolution of drug resistance associated with secondary kinase domain (KD) mutations is the best characterized mechanism of acquired resistance to effective tyrosine kinase inhibitor (TKI) therapy. Medicinal chemistry efforts have largely been devoted toward synthesizing type II TKIs that, by targeting an inactive kinase conformation, are believed to afford greater selectivity than type I TKIs that bind an active kinase conformation. The only previously described TKI with the ability to successfully suppress all resistance-conferring KD mutants (i.e. “pan-kinase” inhibitor) is the type II multikinase TKI ponatinib. Here, we demonstrate that a type I TKI can be potent, selective, and invulnerable to resistance-conferring KD mutation as a mechanism of resistance. Efforts to develop potent, selective type I pan-kinase inhibitors are warranted. Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3–internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3–mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1–2 study

Background Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations are common in acute myeloid leukemia (AML) and are associated with rapid relapse and short survival. In relapsed/refractory (R/R) AML, the clinical benefit of FLT3 inhibitors has been limited by rapid generation of resistance mutations, especially FLT3-D835. Gilteritinib is a potent, highly selective oral FLT3/AXL inhibitor with preclinical activity against FLT3-ITD and FLT3-D835 mutations. The aim of this Phase 1/2 study was to assess the safety, tolerability, and pharmacokinetic (PK) effects of gilteritinib in FLT3 mutation-positive (FLT3mut+) R/R AML. Methods This ongoing pharmacodynamic-driven Phase 1/2 trial (NCT02014558) enrolled subjects from October 2013 to August 2015 who were aged ≥18 years and were either refractory to induction therapy or had relapsed after achieving remission with prior therapy. Subjects were enrolled in one of seven dose-escalation or dose-expansion cohorts that were assigned to receive once-daily doses of oral gilteritinib (20, 40, 80, 120, 200, 300, or 450 mg). Cohort expansion was based on safety/tolerability, FLT3 inhibition in correlative assays, and antileukemic activity; the 120 and 200 mg dose cohorts were further expanded to include FLT3mut+ patients only. Safety and tolerability, and PK effects were the primary endpoints; antileukemic response was the main secondary endpoint. Safety and tolerability were assessed by monitoring dose-limiting toxicities and treatment-emergent adverse events, and safety assessments (eg, clinical laboratory evaluations, electrocardiograms) in the Safety Analysis Set. Findings A total of 252 adults with R/R AML, including 58 with wild-type FLT3 and 194 with FLT3 mutations (FLT3-ITD, n=162; FLT3-D835, n=16; FLT3-ITD and -D835, n=13; other, n=3), received oral gilteritinib (20–450 mg) once daily in one of seven dose-escalation (n=23) or dose-expansion (n=229) cohorts. Gilteritinib was well tolerated in this heavily pretreated population; Grade 3 diarrhea and hepatic transaminase elevation limited dosing above 300 mg/d. The most common Grade 3/4 adverse events were febrile neutropenia (39%; n=97/252), anemia (24%; n=61/252), thromobocytopenia (13%; n=33/252), sepsis (11%; n=28/252), and pneumonia (11%; n=27/252). Serious adverse events in ≥5% of patients were febrile neutropenia (31%; n=78/252), progressive disease (17%; n=43/252), sepsis (14%; n=36/252), pneumonia (11%; n=27/252), and acute renal failure (10%; n=25/252), pyrexia (8%; n=21/252), bacteremia (6%; n=14/252), and respiratory failure (6%; n=14/252). Gilteritinib demonstrated consistent, potent inhibition of FLT3 phosphorylation at doses ≥80 mg/d in correlative assays. While responses were observed across all dose levels regardless of FLT3 mutation status (overall response rate [ORR]=40%), response rate was improved in FLT3mut+ patients at doses ≥80 mg/d (ORR=52%). Among patients with FLT3-ITD, the additional presence of FLT3-D835 did not alter response rate; patients with only FLT3-D835 responded less frequently. Interpretation Gilteritinib had a favorable safety profile and generated potent FLT3 inhibition leading to high rates of antileukemic responses in patients with FLT3mut+ R/R AML. These findings confirm that FLT3 is a high-value target in R/R AML and that long-term success of therapeutic FLT3 inhibition in AML is optimized by agents with potent, selective, and sustained activity against FLT3-ITD mutations and FLT3 tyrosine kinase domain mutations. Funding This study was funded by Astellas Pharma, Inc., by a National Cancer Institute Leukemia Specialized Program of Research Excellence grant (CA100632) awarded to Drs Mark Levis and Jorge Cortes, and by Associazione Italiana Ricerca sul Cancro awarded to Professor Giovanni Martinelli.

FLT3 D835 mutations confer differential resistance to type II FLT3 inhibitors

Activating mutations in FLT3 occur in ~30% of adult acute myeloid leukemia, primarily consisting of internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (~5%), commonly at the activation loop residue D835. Secondary kinase domain mutations in FLT3-ITD, particularly at the D835 residue are frequently associated with acquired clinical resistance to effective FLT3 tyrosine kinase inhibitors (TKIs). Molecular docking studies have suggested that D835 mutations primarily confer resistance by stabilizing an active Asp-Phe-Gly in (‘DFG-in’) kinase conformation unfavorable to the binding of type II FLT3 TKIs, which target a ‘DFG-out’ inactive conformation. We profiled the activity of active type II FLT3 TKIs against D835 kinase domain mutants that have been clinically detected to date. We found that type II inhibitors (quizartinib, sorafenib, ponatinib and PLX3397) retain activity against specific D835 substitutions. Modeling studies suggest that bulky hydrophobic substitutions (D835Y/V/I/F) at this residue are particularly resistant, whereas mutations that preserve interactions between D835 and S838 are relatively sensitive (D835E/N). All mutants retain sensitivity to the type I inhibitor crenolanib. These results suggest that patients with relatively sensitive D835 mutations should be included in clinical trials of type II FLT3 TKIs.

Phase I evaluation of XL019, an oral, potent, and selective JAK2 inhibitor

This phase I study evaluated selective JAK2 inhibitor XL019 in 30 patients with myelofibrosis. The initial dose cohorts were 100, 200, and 300 mg orally on days 1-21 of a 28-day cycle. Central and/or peripheral neurotoxicity developed in all patients. Subsequently, patients were treated on lower doses; neurotoxicity was again observed, leading to study termination. Peripheral neuropathy resolved in 50%, and central neurotoxicity in all patients within months after therapy cessation. Myelosuppression was minimal. The terminal half-life of XL019 was approximately 21 h, with steady state reached by Day 8. International Working Group defined responses were seen in three (10%) patients.

Cost-effectiveness of private umbilical cord blood banking.

OBJECTIVE: To investigate the cost-effectiveness of private umbilical cord blood banking. METHODS: A decision-analytic model was designed comparing private umbilical cord blood banking with no umbilical cord blood banking. Baseline assumptions included a cost of

FLT3 inhibitors in AML: are we there yet?

3,620 for umbilical cord blood banking and storage for 20 years, a 0.04% chance of requiring an autologous stem cell transplant, a 0.07% chance of a sibling requiring an allogenic stem cell transplant, and a 50% reduction in risk of graft-versus-host disease if a sibling uses banked umbilical cord blood. RESULTS: Private cord blood banking is not cost-effective because it cost an additional

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis

1,374,246 per life-year gained. In sensitivity analysis, if the cost of umbilical cord blood banking is less than

The role of kinase inhibitors in the treatment of patients with acute myeloid leukemia.

262 or the likelihood of a child needing a stem cell transplant is greater than 1 in 110, private umbilical cord blood banking becomes cost-effective. CONCLUSION: Currently, private umbilical cord blood banking is cost-effective only for children with a very high likelihood of needing a stem cell transplant. Patients considering private blood banking should be informed of the remote likelihood that a unit will be used for a child or another family member. LEVEL OF EVIDENCE: III