Lauren E. Damon

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.

Increased CD62e+ Endothelial Microparticle Levels Predict Poor Outcome in Pulmonary Hypertension Patients

BACKGROUND Endothelial and leukocytes-derived microparticles (EMPs and LMPs, respectively) are increased in patients with pulmonary hypertension (PH). We hypothesized that the levels of circulating EMPs and LMPs could predict outcome in these patients. METHODS Patients undergoing right heart catheterization for untreated pre-capillary PH were eligible for the study. Baseline hemodynamics and biologic and clinical parameters were measured at the time of enrollment. Measurements of CD62e(+), CD144(+) and CD31(+)/CD41(-) EMPs and CD45(+) LMPs were performed using flow cytometry in venous platelet-free plasma samples. After inclusion, patients were treated at the discretion of the physician and prospectively followed for 12 months. The primary end-point was the combined occurrence of death and re-admission for right heart failure (RHF) or worsening of RHF symptoms. RESULTS Seven of 21 patients (mean age 54.1 +/- 3.5 years, 62% female) experienced the primary end-point during the study period. These patients had higher baseline levels of CD62e(+) EMPs, LMPs and hsCRP (high sensitivity C-reactive protein) compared to patients without events (p < 0.05), whereas no difference was observed for other microparticles and functional and hemodynamics parameters. Receiver operating curve analysis showed that baseline CD62e(+) EMPs levels of >353 events/microl predicted clinical complications. Kaplan-Meier analysis revealed that patients with baseline CD62e(+) EMPs above this cut-off value had a significantly worse prognosis compared with those subjects who had levels below this cut-off (p = 0.02, log-rank statistics). CONCLUSIONS Elevated levels of circulating CD62e(+) EMPs but not LMPs in PH patients prior to treatment are associated with adverse clinical events. Assessment of CD62e(+) EMPs levels may represent a new tool for stratification of PH patients.

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.

Abstract 4737: Saturation mutagenesis of FLT3-ITD: AC220-resistance-conferring kinase domain mutations are restricted to a limited number of residues and are cross-resistant to sorafenib in vitro

Activating mutations in FLT3 are detected in approximately 30 percent of adult acute myeloid leukemia (AML) cases, most commonly involving in-tandem duplication (ITD) events. AC220 is a promising potent and selective inhibitor of FLT3 that has notably achieved morphologic bone marrow remissions in a substantial proportion of relapsed and refractory AML patients in a previous phase I study (Cortes et al, ASH 2009). The ability of AC220 to achieve deep remissions in AML patients suggests that FLT3 activity is essential for survival of malignant myeloblasts in some cases. Although some responses observed in phase I have been durable, many patients ultimately suffered relapsed disease. AC220 is currently being evaluated in a multinational phase II study of AML patients who harbor FLT3 ITD mutations and have failed prior chemotherapy. As has been previously observed with BCR-ABL kinase inhibitors, it is anticipated that loss of response to AC220 may occur through selection of leukemic cells that harbor resistant kinase domain mutations in FLT3. In an effort to identify substitutions in FLT3 that are capable of conferring AC220 resistance in vitro, we have employed a well-validated in vitro mutagenesis approach (Azam et al, Cell, 2003) of FLT3-ITD using the DNA repair deficient E. coli strain XL1-Red, with subsequent selection for resistance in Ba/F3 cells grown in soft agar (devoid of IL-3) containing 20 nM AC220 (approximately 20x the inhibitory concentration 50 (IC50) of unmutagenized FLT3-ITD). Through sequence analysis of over 100 resistant clones, we have identified at least two independent isolates of substitutions occurring at four individual residues scattered throughout the kinase domain. The most commonly isolated mutations involved the “gatekeeper” residue F691 and activation loop residues D835 and Y842, which have also been implicated in preclinical resistance to other FLT3 inhibitors. Each of the kinase domain mutations is sufficient to confer AC220-resistant growth when re-introduced into Ba/F3 cells. Western immunoblot analysis reveals that these mutations confer varying degrees of biochemical resistance to AC220 that correlate with the degrees of resistance observed in cell growth assays. Notably, AC220-resistant mutations FLT3-ITD kinase domain confer in vitro cross-resistance to sorafenib, which has recently been reported to harbor activity in a small number of FLT3-ITD AML cases assessed. Sequence analysis of the FLT3 kinase domain in relapsing AML patients will be required to ascertain the importance of kinase domain mutation in clinical resistance, and is presently ongoing. These studies may inform the development of novel FLT3 inhibitors capable of treating AML that has acquired resistance to AC220 or sorafenib through selection of resistant FLT3 kinase domain mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2011-4737