Quizartinib (AC220) is a potent second generation class III tyrosine kinase inhibitor that displays a distinct inhibition profile against mutant-FLT3, -PDGFRA and -KIT isoforms

Gain-of-function mutations of the FLT3, KIT and PDGFR class III receptor tyrosine kinases (RTK) play important roles as oncogenesis-driving events in several hematologic malignancies. For example, FLT3 mutations are predominantly found in AML associating with a poor prognosis [1‐4], but are also reported in (pediatric) acute lymphoblastic leukemia (ALL) [5]. KIT mutations occur in the vast majority of systemic mastocytosis (SM) [6] and subsets of acute leukemia, including core-binding factor (CBF) [7] and pediatric [8] AML. Certain FLT3 and KIT mutations correlate with inferior outcome in adult AML [4, 9, 10].

PDGFR mutations are frequently found in myeloproliferative disorders, such as Philadelphia chromosome-negative chronic myeloid leukemia (CML), where PDGFR alpha or beta fuses with another gene allowing autoactivation of the tyrosine kinase. Several fusion partners have been described, including FIP1L1 leading to the FIP1L1-PDGFRA fusion gene. This translocation has been associated with hypereosinophilic syndromes and mastocytosis with eosinophilia [11‐13].

Numerous tyrosine kinase inhibitors have been developed to target class III RTKs (see also Discussion). These TKIs have a variable spectrum of activity against different class III RTKs and against various mutant isoforms of these kinases. To date, translation from bench to bedside has resulted in only modest or short-lived effectiveness of these inhibitors in most entities [14‐23] and only a few agents have achieved FDA-approval for selected indications such as CML and HES. With the exception of Ph+ALL, no TKIs have been approved for treatment of acute leukemia so far.

Quizartinib is a novel second generation class III receptor tyrosine kinase inhibitor with superior pharmaceutical properties and an excellent pharmacokinetic profile compared to other agents. Quizartinib was demonstrated to have high efficacy and tolerability in tumor xenograft models that express a FLT3 ITD mutant kinase [24, 25].

A previous study used recombinant enzyme in in vitro kinase assays to identify that quizartinib targets related class III RTKs, such as wildtype and gain-of-function mutant KIT and PDGFR isoforms [24].

Using several cell based assays, we now show, that quizartinib treatment of leukemic cells leads to inhibition of mutant KIT, PDGFR and FLT3 isoforms - with resultant inhibition of cellular proliferation and induction of apoptosis. These effects are seen in vitro as well as ex vivo (primary leukemic blasts). Importantly, potent antitumor activity was seen against distinct (mutated) kinase isoforms, including FIP1L1-PDGFRA, and FLT3 ITD, FLT3 TKD1 and FLT3 TKD2 mutations. Whereas some mutant-KIT and –FLT3 isoforms were sensitive to quizartinib treatment, some mutations such as FLT3 D835V and the most prevalent KIT gain-of-function mutation detected in CBF AML, KIT D816V, was relatively insensitive with regard to quizartinib treatment.

Quizartinib is currently under clinical investigation in FLT3 ITD and wildtype AML. Our data suggests that quizartinib may be an attractive agent for clinical investigation in other settings as outlined here. This would not include the group of mutant-KIT CBF AML that have KIT D816V mutations. However, patients with CBF AML with KIT D816Y or exon 11 mutations or patients with solid tumors associated with KIT and PDGFR mutations, such as GIST might benefit from this agent. Clinical mutation analysis could help identify individuals that are the most likely to respond to quizartinib.

Tyrosine kinase inhibitors are rapidly entering into the clinic. These agents are subject of intensive clinical investigation for the treatment of acute leukemia. For example the following inhibitors are under investigation for mutant ABL1, FLT3 or KIT-associated subtypes (clinicaltrials.gov): sorafenib (e.g. AML: NCT00217646, NCT00373373), sunitinib (e.g. AML: NCT00783653), dasatinib (e.g. CBF-AML: NCT00850382; SM: NCT00979160; Ph + ALL: NCT00103701, NCT00940524), imatinib (e.g. AML: NCT00707408, NCT00744081; activated RTKs/various tumors: NCT00171912), lestaurtinib (e.g. AML: NCT00030186, NCT00079482), tandutinib (e.g. AML: NCT00064584, NCT00274248), masitinib (e.g. SM: NCT00814073) and nilotinib (e.g. AML: NCT01222143; SM/HES: NCT00109707).

Quizartinib (formerly AC220), a N-(5-tert-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo [2,1-b] [1, 3] benzothiazol 2-yl]phenyl}urea dihydrochloride, is a novel class III tyrosine kinase inhibitor with promising in vitro as well as in vivo activity against FLT3 wildtype and mutant isoforms [24]. Compared to other tyrosine kinase inhibitors in evaluation for the treatment of acute leukemia subtypes, quizartinib provides superior bioavailability with longer and higher plasma concentrations achieved in vivo[25] thereby targeting and suppressing the activated kinases more effectively.

We now report that quizartinib not only targets FLT3 wildtype and ITD mutant kinases [24] – but also potently inhibits cellular proliferation and induces apoptosis of cells expressing a broad range of other clinically relevant class III (mutant) RTK isoforms associated with various diseases, including pediatric and adult leukemia (FLT3 and KIT), GIST, seminoma and melanoma (KIT), as well as myeloproliferative neoplasms associated with eosinophilia (PDGFRA). Quizartinib-responsive mutations were thereby detected in the juxtamembrane (ITDs), but also the tyrosine kinase domains (TKD I - K663Q, TKD II - D835Y) of FLT3, as well as in the KIT JM- (V560G) and TK-domains (D816Y). However, as with all tyrosine kinase inhibitors, quizartinib has a distinct potency profile against different autoactivating mutant RTK isoforms - and some mutations in the TKD of FLT3 (D835V, B1-sheet ITDs) and KIT (D816V) proved to be resistant towards quizartinib. Resistancy of FLT3 ITD mutations located in the beta-1 sheet of the first TKD has previously been shown for other TKI [32‐34] as well. In contrast to the BCR-ABL1 fusion transcript demonstrating resistance towards quizartinib, the FIP1L1-PDGFRA fusion mutation revealed extraordinary sensitivity. In CBF AML, which is frequently dependent upon KIT-mediated (gain-of-function) signal transduction, quizartinib demonstrated varying antiproliferative and cytotoxic efficacy in in vitro and ex vivo leukemia cells – in some cases within the low nanomolar range of FLT3 ITD (JM) positive samples. However, the most prevalent KIT mutation in CBF AML, substituting valine for aspartic acid at codon 816 (KIT D816V), was demonstrated to be basically insensitive towards quizartinib in in vitro and ex vivo leukemia cell lines, primary myeloblasts, and in an isogenic Ba/F3 cell model – while substitution of a tyrosine residue (D816Y) retained some sensitivity to quizartinib. Therefore tyrosine kinase genotyping may become a prerequisite for clinical use of this agent.

Moreover, based on our data, we speculate that quizartinib may be a promising agent in solid tumors associated with KIT mutations, such as GIST or melanoma: In addition to a favorable activity against KIT mutant kinases expressed in GIST (and other mutant-KIT neoplasms), the excellent pharmacokinetics with unprecedented achievable plasma concentrations may be advantageous to target bulky solid tumor lesions that have impaired drug uptake. Thus, our data opens new avenues for clinical investigation and further testing of the efficacy of quizartinib in these settings is warranted.

It has to be noted, that IC50s in our studies were coherent in between all cell models used – but higher compared to a previous report [24]. The phenomenon was seen throughout the assays and is therefore most likely due to methodology reasons as we have illustrated with several experimental data:

While individual cell-context specific additional effects (such as additional mutations) can not be fully excluded to have obscured sensitivity profiles in some cell models, methodological differences most likely will account for most of the discrepancies observed: In contrast to previous studies using serum-depleted culture conditions (0.5% FBS), we used serum-repleted medium in all assays (10% FBS for cell lines, 20% for native blasts).

Even more, data from Zarrinkar and colleagues were based on treating refractory/relapsed AML samples [24] – in contrast, samples tested in our assays were isolated from patients with newly diagnosed disease. However, it is believed that refractory/relapsed patient samples have higher sensitivities towards TK-inhibition due to a higher addiction to the respective oncogenic (mutant) tyrosine kinase [37].

To summarize, our findings suggest that quizartinib is a promising agent for treatment of several hematologic and solid human neoplasms. However, due to the quizartinib-specific mutation restricted spectrum of activity, tyrosine kinase mutation screening may be required to identify patients most likely to respond to quizartinib therapy.

Grant support in part by the Deutsche Krebshilfe Foundation (MMS, KKS), the IZKF Program of the Medical Faculty Tübingen (MMS) and the Carreras Scholarship Program (KKS), a grant from the Leukemia and Lymphoma Society (MCH) and a Merit Review Grant from the Department of Veterans Affairs (MCH). We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tübingen University.