Mutations In Tyrosine Kinase Domain Research Articles

Gilteritinib for Relapsed Acute Myeloid Leukaemia with FLT3 Mutation during the COVID-19 Pandemic: Real World Experience from the UK National Health Service

Gilteritinib for Relapsed Acute Myeloid Leukaemia with FLT3 Mutation during the COVID-19 Pandemic: Real World Experience from the UK National Health Service

The FLT3 F691L Gatekeeper Mutation Promotes Clinical Resistance to Gilteritinib + Venetoclax (GILT + VEN) in AML

Background: FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown.Results: Early resistance cell cultures to GILT+VEN were created by exposing MOLM14 cells to GILT 25nM + VEN 25nM alone or supplemented with microenvironmental ligands FGF2 or FLT3 ligand (FL; N=3/group). Media, drugs, and ligands were replenished twice weekly. After 25 weeks, only the cultures exposed to ligand resumed growth (N=1 for FGF2 and N=3 for FL). Ligands were then removed from these early resistant cultures to induce late resistance. There was an initial drop in cell viability but cells resumed growth after only 3.5 weeks (Fig. 1). In contrast, the time to develop early and late resistance to GILT monotherapy was 8 and 15 weeks, respectively. Immunoblot analysis of GILT + VEN early and late resistant cultures demonstrated restoration of FLT3 signaling, as well as phosphorylation of downstream AKT/MAPK pathways. These results also contrasted to late GILT monotherapy resistant cultures, which had downstream AKT/MAPK activation via outgrowth of NRAS mutations. Since FLT3 appeared to be functionally active, we sequenced FLT3 and found that all early and late GILT + VEN resistance cultures had gatekeeper FLT3 F691L mutations. F691L accounted for only in a minority of resistance cultures to GILT monotherapy. To test if FLT3 signaling was important for resistance, we exposed parental cells to higher concentrations of gilteritinib, which have been shown to partly overcome F691L, as well as the FLT3i FF-10101, which binds FLT3 at a different site and is not affected by the F691L mutation. Both of these approaches restored sensitivity to FLT3i in vitro. As expected, the F691L mutation provided broad resistance to most FLT3i (Fig. 2).To validate this mechanism of resistance in patients, we identified a relapsed FLT3-ITD patient who was treated with GILT monotherapy for 5 months, followed by GILT + HMA for 4 cycles, and then GILT + VEN for resistant proliferative disease. After an initial response to GILT + VEN, the leukemia cells began to increase again in the peripheral blood. A repeat genetic test was ordered and the patient was found to have developed a FLT3 F691L mutation at a high variant allele frequency (Fig. 3).Conclusion: We have developed a robust cell line model of early and late resistance to FLT3i that mimics the timing and expansion of resistance mutations in the clinic. Our model of early and late resistance to GILT combinations can prospectively predict mechanisms of resistance. Although uncommon as a mechanism of resistance to GILT monotherapy, our model and early patient data predicts that F691L mutations are more important for GILT + VEN resistance. [Display omitted] DisclosuresTyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Hypomethylating Agent (HMA) Therapy and Venetoclax (VEN) with FLT3 Inhibitor “Triplet” Therapy Is Highly Active in Older/Unfit Patients with FLT3 Mutated AML

Hypomethylating Agent (HMA) Therapy and Venetoclax (VEN) with FLT3 Inhibitor “Triplet” Therapy Is Highly Active in Older/Unfit Patients with FLT3 Mutated AML

A Phase 1, Dose-Escalation Study of Gilteritinib Combined with Chemotherapy in Patients Aged 6 Months to

Background: Acute myeloid leukemia (AML) accounts for ~18% of all childhood leukemias (Puumala SE, et al. Pediatr Blood Cancer. 2013;60(5):728-733). Gilteritinib, an oral FMS-like tyrosine kinase 3 (FLT3) inhibitor, has demonstrated efficacy with favorable tolerability in clinical trials of adults with FLT3-mutated relapsed or refractory (R/R) AML, including internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations (Perl AE, et al. N Engl J Med. 2019;381(18):1728-1740), leading to approval for the treatment of adults with R/R AML in the United States and many other countries/regions. Since the clinical progression of FLT3 ITD AML appears to be similar in children and adults, it is expected that the clinical benefit of gilteritinib in pediatric patients with FLT3 ITD R/R AML should be similar to that demonstrated in adults. We describe an ongoing phase 1, multicenter, open-label, single-arm, dose-escalation study (ClinicalTrials.gov identifier: NCT04240002) investigating the combination of gilteritinib with chemotherapy in children, adolescents, and young adults with FLT3 ITD R/R AML, which will inform the dose for the phase 2 dose-expansion portion of the study.Study Design and Methods: This phase 1 dose-escalation study will enroll patients 6 months to <21 years of age with FLT3 ITD and/or TKD-mutated AML (determined using institutional assay). Key inclusion and exclusion criteria include patients who are in first or greater relapse or are refractory to induction therapy with no more than 1 attempt at remission induction and (a) fully recovered from the acute toxic effects of all prior therapy, (b) have a Karnofsky (if ≥16 years) or Lansky (if <16 years) score ≥50, and (c) been ≥90 days relapsed since hematopoietic stem cell transplantation with no active graft-versus-host disease, if relevant. Patients will undergo screening, including complete physical exam, subject-reported symptoms, and areas of disease (Figure). Patients will be enrolled in 1 of the 3 following groups: group 1 (2 to <21 years), group 2 (1 to <2 years), and group 3 (6 months to <1 year). Gilteritinib will be administered once daily starting from day 8 through day 21 of a 28-day cycle at the starting dose of 2 mg/kg/day for Group 1 and 1 mg/kg/day for Groups 2 and 3, with escalation to 2 or 3 mg/kg/day (the latter to be evaluated only if there is lack of toxicity or acceptable dose-limiting toxicity [DLT] and lack of sufficient gilteritinib activity observed at 2 mg/kg/day). Patients in all groups will receive FLAG (fludarabine, 30 mg/m 2/day; cytarabine, 2000 mg/m 2/day; granulocyte-colony stimulating factor, 5 μg/kg/day) on days −1 to 5 and prophylactic single intrathecal cytarabine (age-adjusted dose). Patients who complete 2 cycles of treatment will have the option to participate in long-term treatment (LTT) with gilteritinib for ≤2 years. An end of treatment (EOT) visit will occur 7 days after the last dose of gilteritinib or before initiation of another anticancer therapy. A follow-up visit will be performed 28 days after the EOT visit in patients who are ineligible for LTT. Patients eligible for LTT will undergo a follow-up visit 28 days after the LTT EOT visit. Follow-up for survival will be performed every 3 months for ≤2 years from the 28-day follow-up visit.Study End Points: The primary end point is identification of the maximum tolerated dose and/or recommended phase 2 dose based on the DLT observed in treatment cycle 1 and biologic activity according to plasma inhibitory activity (PIA). The secondary end points are inhibition of phosphorylated FLT3, gilteritinib pharmacokinetics, safety, tolerability, toxicity, event-free survival, overall survival, minimal residual disease assessment, and acceptability and palatability assessment of the formulation. The exploratory end points are correlation of clinical responses to gilteritinib with FLT3 PIA levels, correlation of FLT3 PIA levels and clinical responses to gilteritinib therapy with FLT3 ligand levels before and after exposure to FLAG chemotherapy, and mechanisms of innate and acquired resistance to gilteritinib. All data will be summarized with descriptive statistics for continuous end points and frequency/percentage for categorical end points. [Display omitted] DisclosuresConnor: Astellas Pharma Inc.: Research Funding. Fan: Astellas Pharma Global Development: Current Employment. Gill: Astellas Pharma Global Development: Current Employment. Hill: Astellas Pharma Global Development: Current Employment; Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder. Philipose: Astellas Pharma Global Development: Current Employment. Delgado: Astellas Pharma Global Development: Current Employment. Tiu: Astellas Pharma, Inc.: Current Employment. Reinhardt: Abbvie: Other: Advisory board; Eusa: Other: Advisory board; Astellas Pharma Inc.: Research Funding; Janssen: Other: Advisory board; BluebirdBio: Other: Advisory board; BMS: Other: Advisory board; Novartis: Other: Advisory board; JAZZ: Other: Advisory board. OffLabel Disclosure:New Indication

Onset of Blast Crises in CML Patients in Treatment-Free Remission: Descriptive Analysis of 4 Cases

Aims: The onset of blast crisis (BC) in initially chronic phase (CP) CML patients that entered treatment-free remission (TFR) after TKI is an exceptional event, however, there is emerging evidence that this may occur in such patients (pts), although the pathogenesis remains unclear to date.Methods: Anonymous clinical case retrospective data collection from patients' datafiles, after written agreement of living patients, centralisation of available frozen nucleic acid collection from diagnosis and from blast crisis and reanalysis by Next-Generation Sequencing of samples (ASXL1, ASXL2, BCOR, CALR, CBL, CEBP alpha , CSF3R, DNMT3A, EP300, ETNK1, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2 JAK2, KIT, , KRAS,MPL, NPM1, NRAS, PHF6, PTPN11 , RAD2, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG1, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2 genes analysed) on Illumina platform. CNV analysis were performed using VisCAp or in-house pipelines and/or by Multiplex Ligation Dependant Probe Amplification (MLPA). ABL1 tyrosine kinase domain mutations were screened by NGS on cDNA or directly on DNA. BCR-ABL1 transcripts are expressed in % (IS) with at least 32,000 copies of ABL1 as control. All patients discontinued their TKI after 2 years of MR4.5 and a TKI was resumed in case of MMR loss.Results: Along 15-year experience of TFR in our country and ≥ ~800 patients experiencing a TKI cessation attempt, informations from 4 (~0.5%) TFR CML patients entering BC have been collected. All patients harboured Major BCR transcripts.The chronic phase characteristics are mentioned in Table 1.All these long-lasting CP CML pts were ELTS risk score low, and 1 was harbouring ACA at diagnosis. One pt was mutated for ASXL2 and 2 mutations for EP300, found again at BC. Three pts had IFN-a prior to imatinib for all. Three out of 4 lost their MMR after a first cessation attempt at 12, 10 and 3 months after cessation. Pt #1 experienced a 2 nd cessation attempt 52 months after re-initiation of TKIs and entered lymphoid BC 6 months after a second resumption of TKI for a 2 nd MMR loss.The BC characteristics are mentioned in Table 2.Three out of 4 BC were lymphoid, one had ACA different from those at CP diagnosis. Two pts explored had multiple mutations in Runx1, U2AF1, EP300 and ASXL2 genes, not present at CP diagnosis, in addition to multiple ABL1 mutations in 2 out of 4 pts (2 T315I, 3 P-Loop mutations). All pts underwent chemotherapy + various TKI leading to complete remission (CR) in all and 3 out of 4 pts could be allotransplanted in CR, one relapsed shortly after transplant, and a second one 34 months after transplant. Overall 3 pts are alive with 1 in controlled relapse.Conclusions: The onset of BC after TFR for sustained deep molecular response remains an exceptional event and is probably not induced by this therapeutic procedure. These 4 cases underline the need for a sustained long-lasting molecular follow-up of pts in TFR, although the majority of these BC seem sudden. [Display omitted] DisclosuresBauduer: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria.

Midostaurin Plus Intensive Chemotherapy for Younger and Older Patients with Acute Myeloid Leukemia and FLT3 Internal Tandem Duplications

Midostaurin Plus Intensive Chemotherapy for Younger and Older Patients with Acute Myeloid Leukemia and FLT3 Internal Tandem Duplications

Investigation of FMS-Like Tyrosine Kinase 3 Mutation Frequency in Myelodysplastic Syndrome

Introduction: FMS-Like Tyrosine Kinase Class 3 (FLT3) mutations harbor poor prognosis, high relapse, and decreased overall survival in acute myeloblastic leukemia (AML). This mutation is also known to be demonstrated in myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia and acute lymphoblastic leukemia. This study included 94 MDS-diagnosed patients and we tried to investigate FLT3 mutation frequency (as tyrosine kinase domain-TKD and internal tandem duplication-ITD).&#x0D; Materials and Methods: Polymerase chain reaction (PCR), restriction fragment length polymorphism, and agarose-gel electrophoresis methods were used to analyze the mutation. The blood samples were collected in K3-EDTA tubes, and total DNA was isolated using genomic DNA isolation kits (GeneMark, Cat No: DP023P). For the detection of FLT3-ITD mutation, PCR was performed to amplify a 330- base pair fragment of exons 11 and 12 of FLT3 using FAM (Carboxyfluorescein)-labeled ITD-11F and HEX (Hexachloro-Fluorescein )-labeled ITD-12R primers in a thermal cycler (Eppendorf). Similarly, to detect D835 mutation, a 115- bp region of exon 17 of the FLT3 gene region was amplified using primers.&#x0D; Results: One patient was found FLT3-ITD positive (1.1%). The patient was 64-year-old and diagnosed with MDS-excess blast type 2 according to the World Health Organisation 2016 myeloid neoplasm classification. He transformed to AML within 19 months and subsequently died after 1 month. No patient with tyrosine kinase domain mutation was detected.&#x0D; Conclusion: FLT3 mutation is considered a significant parameter to define prognosis in AML. The routine workup of FLT3 screening and the potential of targeting FLT3 inhibition for high-risk MDS may be taken into consideration in the future.

Tyrosine kinase domain mutations in chronic myelogenous leukemia patients: A single center experience.

Introduction:Despite the impressive responses achieved with tyrosine kinase inhibitor (TKI) therapy, treatment resistance develops in 16–33% of patients of chronic myelogenous leukemia (CML). Of the BCR-ABL1 dependent mechanisms, mutations in the tyrosine kinase domain (TKD) are the commonest cause of resistance.Material and Methods:Allele specific oligonucleotide - polymerase chain reaction (ASO-PCR) was done for testing the six common TKD mutations, T315I, G250E, E255K, M244V, M351T, and Y253F.Results and Conclusion:TKD mutation study was done on 83 patients. Of these 44 (53%) were positive for one or more mutations. On analyzing specific mutations, E255K was the commonest mutation seen in 24 (29%) cases, followed by T315I in 23(28%) cases. Y253F mutation was not seen in the present study sample. In the present cohort of 83 patients, 29 (35%) cases were positive for single mutation, 12 (14%) had two mutations and 3 (4%) had three mutations.

AML-162: Efficacy and Safety of Venetoclax in Combination with Gilteritinib for Relapsed/Refractory FLT3-Mutated Acute Myeloid Leukemia: Updated Analyses of a Phase 1b Study

<h3>Background/Objective:</h3> To provide updated results from a Phase 1b trial (NCT03625505) of BCL-2 inhibitor venetoclax and FMS-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib (Ven+Gilt) in patients with relapsed/refractory (R/R) FLT3-mutated (<i>FLT3^Mut</i>⁺) acute myeloid leukemia (AML). <h3>Methods:</h3> Patients with R/R AML <i>FLT3^Mut</i>⁺ received up to Ven 400mg daily with Gilt (80 or 120 mg) daily in 28-day cycles, following Ven ramp-up. Primary endpoint was modified composite complete remission (mCRc) to align with Phase 3 ADMIRAL trial CRc responses. Secondary endpoint was mCRc duration of response (DOR); overall survival (OS), and changes in <i>FLT3</i> allelic burden were exploratory. Adverse events (AEs) were monitored. <h3>Results:</h3> At data cutoff (November 30, 2020), 43 patients (median age 63 yrs) with <i>FLT3^Mut</i>⁺ were enrolled. <i>FLT3</i> internal tandem duplications (ITD) were identified in 86% patients, and 14% had only tyrosine kinase domain (TKD) mutations. Baseline cytogenetic risk was favorable in 5%, 55% intermediate, 31% poor, and 12% had no mitoses/missing data. Median (range) prior lines of therapy were 2 (1–5), and 77% patients had 2 or more prior lines of therapy: 65% received at least one prior FLT3 inhibitor, and 7% received prior Ven; 33% had prior transplant. Grade 3/4 AEs were reported in 98% of patients; grade ≥3 cytopenias occurred in 79% and were predominantly managed by Ven/Gilt dose interruptions. The only grade 3/4 nonhematologic AEs reported in >20% of patients was pneumonia (n=9; 21%) and 1 instance of clinical tumor lysis syndrome. Serious AEs were reported in 74% of patients. Overall, 30- and 60-day mortality rates were 0% and 12%, respectively. AEs led to either Ven or Gilt interruptions in 56% patients, reductions in 7%, and discontinuations in 14%. mCRc was achieved by 86% of <i>FLT3^Mut</i>⁺ efficacy population (36/42) with median time to first response of 1.0 mo (range: 0.7–4.6) and 86% with prior FLT3 tyrosine kinase inhibitor (TKI) exposure (24/28). FLT3 molecular clearance (<10^–2) was observed in 69% of mCRc patients with PCR analyzed at baseline and at least 1 follow-up timepoint (18/26). <h3>Conclusions:</h3> Updated analyses show Ven+Gilt achieved high rates of mCRc in patients with heavily pre-treated and prior TKI-exposed R/R <i>FLT3^Mut</i>⁺AML with encouraging molecular clearance rates. Using similar criteria to previous <i>FLT3^Mut</i>⁺ studies, high mCRc rate with Ven+Gilt suggests strong anti-leukemic activity. Cytopenias were prominent but manageable.

Abstract 1257: HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors

Abstract Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduces overall survival. Approximately 30 % of AML patients carry FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutations. Sustained FLT3 inhibition can result in the emergence of resistance-conferring genetic alteration in TKD domain, usually at residues D835 and F691. Therefore, acquired TKD mutations has become a critical therapeutic target in AML therapy. On the other hand, SYK is one of kinases deeply implicated in many hematologic malignancies and highly activated in FLT3 mutation AML. SYK overexpression is known to promote over transformation of FLT3 driven AML and induce resistance to FLT3 targeted therapy. In this study, we characterized HM43239, a novel FLT3 inhibitor with SYK inhibitory activity, and assessed its potential as a novel therapeutic agent to overcome the resistances against AML therapy using current FLT3 inhibitors. HM43239 is an orally active small molecule inhibitor and it exhibited sub-nanomolar potency on binding affinity for FLT3 wild type, ITD, TKDs and ITD/TKDs mutants. HM43239 potently inhibited phosphorylation of FLT3 and its downstream such as p-STAT3/p-STAT5 dose dependently in both MOLM-14 cells harboring FLT3 ITD/F691L and FLT3 ITD/D835Y. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. Moreover, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG-1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Furthermore, HM43239 showed to be exhibited good inhibitory activity against FLT3 mutated AML cell lines and effectively regress the tumors in MOLM-14 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. On the other hand, it was confirmed that HM43239 strongly inhibited the phosphorylation of SYK in the medium of co-culture of stromal cells and AML cells. As a result, HM43239 alone more effectively induced tumor regression and prolonged the survival duration of animals than an approved FLT3 inhibitor (e.g. gilteritinib) in resistant FLT3 ITD/D835Y or ITD/F691L mutated MOLM-14 xenograft mice models. These results suggest that HM43239 could overcome the resistance induced by bone marrow microenvironment in AML patients. Taken together, HM43239 showed strong anticancer activity through various in vitro and in vivo preclinical models of AML, implicating the mechanism of overcoming resistance and preventing relapse. The effect of HM43239 in human would be demonstrated in ongoing Phase I/II clinical trials (NCT03850574) to develop promising therapeutics for patients with AML. Citation Format: Inhwan Bae, Jaeyul Choi, Jiyoung Song, Joo-Yun Byun, Eunyoung Lee, Taehun Song, Yu-Yon Kim, Yesol Bak, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1257.

Abstract 1428: PARP inhibitor resensitizes TKI-resistant AML to TKI

Abstract Internal tandem duplications within the juxtamembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) are present in approximately 25% of patients with acute myeloid leukemia (AML) and is an independent predictor of poor prognosis. FLT3-ITD is a constitutively active form of the receptor and aberrantly signals through signal transducer and activator of transcription (STAT) 5 which feeds-back to stabilize FLT3 through the downstream effector, Pim-1 kinase. FLT3-specific tyrosine kinase inhibitors (TKI) have clinical effects in FLT3-ITD AML but responses are limited due to the development of resistance through various means, including point mutations in the FLT3 tyrosine kinase domain commonly at D835 in the activation loop and F691 in the gate-keeper residue. Novel therapeutic strategies are therefore required for overcoming TKI-resistance. Poly (ADP-ribose) polymerase (PARP) 1 functions by poly-ADP-ribosylating (PAR) itself and other proteins primarily to catalyze DNA repair, however, PARP can also regulate the activity and function of proteins involved in other various processes. Notably, PARP inhibitors (PARPi) are novel cancer therapeutics that induce synthetic lethality in cancers with BRCA mutations. Recent studies also show that concurrent treatment of PARPi and TKI can effectively induce synthetic lethality in BRCA-proficient TKI-sensitive FLT3-ITD AML. However, the effects of PARPi in TKI-resistant disease has yet to be explored. We previously reported elevated reactive oxygen species (ROS) in FLT3-ITD AML leading to upregulated PARP1 and we now show that, compared to parental TKI-sensitive Molm14 cells, D835Y and F691L point-mutated TKI-resistant forms have significantly increased ROS production, and subsequently increased DNA damage and PARP1 protein expression. Interestingly, TKI-resistant cells also have elevated phospho-STAT5 (pSTAT5) and subsequent CRISPR-Cas9 knockout of PARP1 leads to reduction in pSTAT5 as well as the downstream targets, Pim-1 and FLT3. Significantly, STAT5 protein was found to immunoprecipitate with PAR and PARP1, suggesting for the first time that PARP1 may play a direct role in regulating STAT5. Finally, we have found that low doses (5-50nM) of the potent PARPi, Talazoparib (Tal), in combination with low dose (10nM) of the TKIs, Quizartinib (Quiz) or Gilteritinib (Gilt), is synergistically lethal in the otherwise TKI-resistant cells. Additionally, sequential treatment of TKI-resistant cells with Tal (72hr) prior to treatment with Quiz (72hr) or Gilt (72hr) was still synergistic whereas the reverse is not. This suggests a novel model whereby Tal may resensitize cells to TKI by decreasing PARP1 effects on pSTAT5 activity which feeds back to reduce FLT3 receptor activation. Therefore, this combination has potential for treatment of TKI-resistant AML and studies are underway to translate these findings with the intent of developing an effective therapy for patients with this disease. Citation Format: Anna J. Dellomo, Rena G. Lapidus, Mariusz Karbowski, Maria R. Baer, Tami J. Kingsbury, Feyruz V. Rassool. PARP inhibitor resensitizes TKI-resistant AML to TKI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1428.

Abstract 1050: Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is the most common and aggressive acute leukemia in adults, with a 25% 5-year survival rate. FLT3 is the most frequently mutated gene in AML. About 25% of AML patients harbor FLT3 internal tandem duplication (ITD) mutations, and about 8 % of patients harbor FLT3 tyrosine kinase domain (TKD) mutations. Both FLT3-ITD and FLT3-TKD mutations constitutively activate the protein, which causes poor survival. Gilteritinib is a highly potent and selective oral FLT3 inhibitor recently approved by the FDA. Although gilteritinib showed strong single agent activity in AML patients with FLT3 mutations, the development of gilteritinib resistance limits the long-term efficacy of this treatment, indicating that combination therapy may be advantageous for AML patients with FLT3 mutations. FLT3 mutations are known to be anti-apoptotic. Myeloid cell leukemia-1 (MCL-1), an anti-apoptotic protein, expressed in a large percentage of the AML patient population, plays a critical role in AML cell survival and drug resistance. AMG 176 is a potent, selective and orally bioavailable MCL-1 inhibitor, which induces rapid commitment to apoptosis in AML. Here we demonstrated that AMG 176 and gilteritinib combination treatment synergistically targeted FLT3-ITD mutated AML. A strong synergistic effect (Combination index &amp;lt;0.2) was observed in two FLT3-ITD mutated AML cell lines treated with the AMG 176 plus gilteritinib combination. Increased Bcl-2-like protein 11 (BIM) expression and decreased BIM/MCL-1 interaction was observed under the combination treatment, coupled with a significant increase in apoptosis. In the MV4-11 AML xenograft model, mice treated with two cycles of AMG 176 (2 days on 5 days off) at 30 mg/kg or with 3 mg/kg gilteritinib daily showed 76% or 74% tumor growth inhibition respectively. A statistically significant increase in efficacy was observed in the combination treatment group, with 92.8% tumor regression, including 50% of mice being tumor free. No significant difference in body weight was found between groups. Our results show that AMG 176 plus gilteritinib treatment was effective and well tolerated in mouse models of AML, which supports further evaluation in a clinical setting. Citation Format: Xiaoyue Chen, Sean Caenepeel, Brian Belmontes, Patricia L. McElroy, Karen Rex, Tao Osgood, Paul Hughes. Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1050.

Comparative Assessment of FLT3 Variant Allele Frequency By Capillary Electrophoresis and Next-Generation Sequencing in FLT3mut+ patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Who Received Gilteritinib Therapy

Comparative Assessment of FLT3 Variant Allele Frequency By Capillary Electrophoresis and Next-Generation Sequencing in FLT3mut+ patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Who Received Gilteritinib Therapy

10-Year Outcome of Chronic Myeloid Leukemia Patients Resistant to Frontline Imatinib

Background: Imatinib remains the most frequently prescribed tyrosine kinase inhibitor (TKI) for patients (pts) newly diagnosed with chronic myeloid leukemia (CML). However, 40-45% patients need to switch to alternative drug because of resistance (20-25%) or intolerance (20%). The mechanism of resistance, although unknown in the majority of cases, is caused by the presence of BCR-ABL1 tyrosine kinase domain (TKD) mutations in approximately 20-30% of pts. Phase II clinical studies of second- and third-generation TKIs (2GTKI and 3GTKI) in imatinib resistance have shown that approximately 50% of pts achieve durable cytogenetic and molecular responses. The differential sensitivities of TKD mutations to the various TKI now allow ‘tailored‘-therapy if mutations are present. We report the long-term outcome of pts who developed imatinib resistance.Methods: From our institutional database we identified pts who failed imatinib at 400mg daily as first-line therapy for CML in chronic phase (CP). Because the definitions of optimal response and resistance have changed over time, we confined our analysis to those pts whose therapy was changed because of resistance and who had TKD mutation testing at the time of resistance. We further stratified pts according to their reverse transcriptase polymerase chain reaction assay (RT-qPCR) at the time of resistance into three groups: group 1: RT-qPCR >0.1% <1%; group 2: 1-10%; group 3: >10%.Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Mayer function and the outcomes between groups were investigated with long-rank test. The OS was calculated from the date of diagnosis till the date of last contact; PFS, defined as progression to accelerated or blastic phases or death without progression, was calculated from the imatinib start date till the date of last contact.Results: 119 pts satisfied our eligibility criteria: baseline characteristics and outcome are shown in the Table 1.The median follow-up from start of imatinib first-line was 110 months (range 18-230). Thirty-one (26%) pts received non-TKI therapy after imatinib failure, of whom 26 (21.8%) received allogeneic transplant. Fifteen pts (12.6%) died (9 of CML progression, 5 due to transplant complications and 1 of mesothelioma). Of the 119 pts, 24 had a TKD mutation (Table 2) at the time of imatinib resistance. Thirteen of 51 pts (25.5%) diagnosed from 2007 onwards had a TKD mutation at time resistance, compared to 11/68 (16.2%) diagnosed between 2000 and 2006 (p=0.25, Chi-squared test). Also, looking at the number of pts mutated at any time during the follow-up, there was no statistically significant difference among the different years of diagnosis (18 out of 51, 35.3%, from 2007 onwards compared to 14 out of 68, 20.9%, before 2007; p= 0.097, Chi-squared test).The 10-year estimated OS and PFS for the entire cohort were 86.2% and 74.5%, respectively. At last follow-up, 85 of 104 (81.7%) surviving pts were in MR3 or deeper response. OS and PFS were not influenced by TKD mutation status, transcript type or year of diagnosis. However, outcome was affected by the level of RT-qPCR at the time of resistance: 10-year OS was 100%, 86.6% and 70.9% for groups 1, 2 and 3 respectively (p = 0.004); 10-year PFS was 97.1%, 86.6% and 70.9% for group 1, 2 and 3 respectively (p < 0.001).In order to understand whether the importance of the molecular response was valid across different treatment periods, we further analysed the cohort after splitting it in pts diagnosed before and after 2007. Interestingly, the difference was no longer significant for pts diagnosed more recently, where the 10-year OS was 100%, 88.9% and 83.1% (p = 0.28) and the 10-year PFS was 90.9%, 90% and 71.1% (p = 0.082), respectively, for each of the above mentioned RT-qPCR groups (Figure).Conclusion: our study has found an unexpected high OS in pts failing imatinib front-line for resistance. Mutational status did not impact OS and PFS. After stratifying pts according to the degree of response to imatinib, it seems that the ready availability of 2GTKI and 3TGKI in the more recent years could be responsible for the positive effect on the outcome, rescuing pts with a higher grade of resistance from progression and death. Finally, our results confirm prior observations that the achievement of complete cytogenetic response on imatinib (RT-qPCR <1%IS) confers an excellent outcome, irrespective of subsequent therapy. [Display omitted] DisclosuresShacham Abulafia:Ariad: Other: recieved grant from ariad. Milojkovic:Novartis: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Incyte: Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Foroni:Incyte: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Apperley:Therakos: Honoraria; Incyte: Honoraria; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses , Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Sun Pharma: Honoraria.

TP-0903, a Novel Axl Inhibitor with Activity in Drug Resistant FLT3-ITD+ AML through a Mechanism That Includes FLT3 Inhibition

Abnormal expression of receptor tyrosine kinase Axl has been associated with poor prognosis in different cancers including acute myeloid leukemia (AML) and has been linked to drug resistance against chemotherapy as well as targeted therapies including FLT3 inhibitors (Park et al. Leukemia 2015). With its role in tumorigenesis, Axl has been actively pursued as a potential therapeutic target with small molecule inhibitors. TP-0903, a type I inhibitor, was designed in silico on an Axl scaffold that does not clash with the Axl gatekeeper residue, Leu620 (Mollard et al. ACS Med Chem Lett 2011). Since Axl inhibitors are known to also interact with FLT3 (Myers et al. J Med Chem 2016), we evaluated the hypothesis that TP-0903 demonstrates preclinical activity against FLT3 -ITD+AML through a mechanism that potentially involves both Axl and FLT3 inhibition.TP-0903 was evaluated against Axl and FLT3 wildtype (WT), ITD and tyrosine kinase domain (TKD) mutations in binding and kinase assays. Inhibition of cell viability was evaluated in Ba/F3 cells transfected with GFP vector control (VC), FLT3 -ITD, or TKD mutations, FLT3 -ITD+ AML cell lines (MOLM13, MOLM13-Res harboring a ITD/D835Y mutation, and MV4-11), and primary diagnostic FLT3 -ITD+ blast samples. Inhibition of FLT3 signaling was determined by Western blot analysis. In vivo activity was evaluated in systemic MOLM13 and MOLM13-Res cell xenograft models in NOD scid gamma (NSG) mice; 1x106 luciferase expressing cells were injected intravenously to female NSG mice, and TP-0903 or vehicle was administered by oral gavage for 3 weeks, starting on day 7-10 post tail vein injection. TP-0903 40-60 mg/kg was administered once daily (qd), and 80 mg/kg was administered on Monday, Wednesday, and Friday (M/W/F). Progression of leukemia was monitored using bioluminescence imaging and Kaplan-Meir analysis was performed for animal survival.TP-0903 had potent binding affinities to Axl, FLT3 WT and all FLT3 mutants evaluated, expressed as Kd: Axl (8.2 nM), FLT3 WT (0.93 nM), ITD (5.6 nM), D835H (1.9 nM), D835V (2.1 nM), D835Y (1.4 nM), ITD/D835Y (0.79 nM), and ITD/F691L (1.9 nM). In a kinase assay, TP-0903 inhibited FLT3 ITD and D835Y with IC50 values of 3.9 nM and 0.12 nM, respectively. TP-0903 inhibited the viability of Ba/F3 transfected with VC (mean IC50 of 3 independent experiments, 47 nM), and activity was more potent against all cells expressing different FLT3 mutants: FLT3 -ITD (15 nM), D835H (16 nM), D835Y (22 nM), ITD/D835H (16 nM), ITD/D835Y (20 nM), and ITD/F691L (16 nM). Western blot analysis of lysates from TP-0903-treated Ba/F3 cells expressing ITD, D835Y, ITD/D835Y, or ITD/F691L mutants showed inhibition of phospho-FLT3 and phospho-STAT5 at 10-100 nM. In FLT3 -ITD+ AML cell lines, TP-0903 demonstrated potent activity with mean IC50 values of 21 nM, 16 nM, and 17 nM in MOLM13, MOLM13-Res, and MV4-11 cells, respectively; the IC50 in primary blast samples ranged from 38-80 nM. In vivo, TP-0903 delayed the outgrowth of MOLM13 cells and significantly prolonged survival in a dose/schedule dependent manner compared to vehicle-treated mice with the following median survival: vehicle (16 days), TP-0903 40 mg/kg qd (26 days, P=0.0039), TP-0903 60 mg/kg qd (32 days, P=0.002), and 80 mg/kg M/W/F (24 days, P=0.0015). In an ongoing MOLM13-Res xenograft model, TP-0903 60 mg/kg qd has suppressed the outgrowth of leukemic cells up to 11 days of treatment compared to vehicle-treated mice (P=0.0002).TP-0903, a novel Axl inhibitor with FLT3 inhibition activity, shows promising in vitro and in vivo activity in de novo and drug resistant FLT3 -ITD+ AML. Given that TP-0903 was initially designed to bind to the gatekeeper leucine residue in Axl, TP-0903 may overcome resistance to the clinically relevant FLT3 gatekeeper F691 substitution mutation to leucine, which most FLT3 inhibitors are vulnerable to. Further in vitro studies to evaluate mechanism of action and in vivo studies for dose optimization and efficacy evaluations in additional models of FLT3 -ITD+ AML are ongoing. DisclosuresWhatcott:Tolero Pharmaceuticals, Inc.: Employment. Warner:Tolero Pharmaceuticals, Inc.: Employment. Blum:Boerhinger Ingelheim: Research Funding; Pfizer: Consultancy; Astellas: Consultancy.

CG '806, a Highly Potent First-in-Class Pan-FLT3/BTK Inhibitor, Targets Multiple Clusters of Kinases Operative in Diverse Subtypes of Acute Myeloid Leukemia

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/ Bruton's Tyrosine Kinase (BTK) multi-kinase inhibitor. This small molecule was designed to inhibit the wild type (WT) and C481S mutant forms of BTK, as well as the WT and mutant forms (including the internal tandem duplication, ITD) of the FLT3 receptor tyrosine kinase. Previously (2017 AACR Hematologic Malignancies abstract # 25 and #44), CG'806 was shown to have nM potency against FLT3 mutation-driven (including ITD and point mutations in tyrosine kinase domain) human acute myeloid leukemia (AML) primary patient cells, and in cultured human (eg. MV4-11 and MOLM-13) and mouse (Ba/F3) AML cell lines. CG'806 inhibited FLT3 signaling, induced apoptosis, and eradicated MV4-11 tumors with no observed toxicity in murine xenograft models. Because AML cells demonstrate extreme heterogeneity in gene expression and because FLT3 mutations account for no more than 30% of AML patients, the effect of CG'806 was further evaluated in AML cells without FLT3 mutations. Immunoblotting demonstrated that CG'806 potently decreased phospho-BTK, phospho-aurora kinases A/B/C and phospho-H3S10 without affecting FLT3 activity in AML cells having low/normal expression levels of homozygous WT FLT3 (eg. KG-1 and NOMO-1). Measurement of BrdU incorporation by flow cytometry demonstrated that CG'806 induced G2/M cell cycle arrest or polyploidy and subsequent apoptosis in FLT3 WT AML cell lines. Interestingly, CG'806 was found to induce G0/G1 cell cycle arrest but not polyploidy in FLT3-ITD driven MV4-11 and MOLM-13 cells with no effect on activity of BTK or aurora kinases at concentrations that completely inhibited FLT3 signaling (FLT3/STAT5/ERK). These findings indicate that the molecular mechanism of cell killing by CG'806 is context-dependent and varies in different AML subtypes. Evaluation of enzyme inhibitory activity (IC50) against 583 kinase enzymes, as well as binding affinities (Kd) with 483 kinase proteins, revealed pM to low nM IC50 and Kd values for CG'806 against the FLT3, BTK, AURK, c-Src, c-Met, c-Ret, DDR, TRK and STE/MAPK families of kinases. The ability of CG'806 to target multiple families or clusters of kinases is responsible for its broad efficacy in different AML subtypes driven by different genomic aberrations. Together, these findings illustrate the ability of CG'806 to inhibit entire FLT3 family of kinases, BTK and the AURK families and pathways that are operative in AML, thereby supporting the development of CG'806 for treatment of patients with FLT3-ITD and other subpopulations of patients with AML. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Mechanisms of Acquired Resistance to Gilteritinib Therapy in Relapsed and Refractory FLT3 -Mutated Acute Myeloid Leukemia

Introduction: A major mechanism of clinical response to FLT3 tyrosine kinase inhibitors (TKIs) is the induction of terminal granulocytic differentiation of leukemic blasts (Sexauer, et al. Blood 2012). However, the clinical activity of many FLT3 TKIs is limited by novel, treatment-emergent activating mutations in FLT3 that confer in vitro resistance–particularly substitutions at D835 in the tyrosine kinase domain (TKD) activation loop and the so-called “gatekeeper” residue F691. Gilteritinib (ASP2215) is a highly potent and selective oral inhibitor of FLT3 that is active against both FLT3 -internal tandem duplication (ITD) and TKD mutations and is currently in phase 3 trials. As a single agent, gilteritinib shows significant clinical activity in relapsed/refractory (R/R) FLT3 -mutated AML (Perl, et al. Lancet Oncol 2017), but mechanisms of therapeutic resistance have yet to be systematically described. We therefore analyzed the serial morphologic and genetic data of patients treated with gilteritinib to better characterize response and identify treatment-emergent genetic alterations associated with disease progression.Methods: We studied adults with R/R FLT3 -mutated AML who were treated at FLT3 inhibitory doses (≥80mg/day) on a phase 1/2 study of gilteritinib at 2 institutions (CHRYSALIS trial, NCT02014558). Clinical and laboratory data were collected from chart review. FLT3 PCR and karyotype were sent at study entry and each treatment assessment, and next generation sequencing (NGS) results were recorded at study entry and completion. FLT3 mutant:wild-type (WT) allelic ratios were converted to mutation frequencies. Subjects were scored as a “differentiation response” if they had a >50% reduction in marrow blasts from baseline with granulocytic hyperplasia and a stable or increased FLT3 mutation frequency, while subjects were scored as a “response without differentiation” if they had a >50% reduction in marrow blasts with at least a 2-fold reduction in FLT3 mutation frequency. All other responses were considered “persistent disease.”Results: 30 subjects were studied. 4 were excluded due to a lack of evaluable data and 5 are currently being analyzed. Of the remaining 21 subjects, the median age was 66 years (range 22 - 87). 19/21 subjects (90%) had a FLT3 -ITD mutation at study enrollment, including 3 (14%) with both FLT3 -ITD and FLT3 -D835 mutations. Two subjects (10%) had a FLT3 -D835 mutation only. 10/21 subjects (48%) had a “differentiation response,” 7/21 subjects (33%) had a “response without differentiation”, and 4/21 subjects (19%) had persistent disease on gilteritinib. The median duration of gilteritinib therapy was 154 days (range 42-536+ days). One subject remains on gilteritinib at the time of data collection and 16 have progressed, with 4 withdrawn for transplant or toxicity. 9/19 subjects (47%) with serial cytogenetic data developed new cytogenetic abnormalities during therapy compared to pre-study karyotypes, including a new BCR-ABL1 fusion at progression in 1 subject. Cytogenetic evolution did not always associate with disease progression. New somatic mutations developed on gilteritinib in 7/16 progressing subjects and 1 non-progressing subject on a pre-transplant marrow (50%), including mutations in NRAS (n=4), IDH2 (n=1), CBL (n=1), and TBL1XR1 (n=1). 3 patients acquired FLT3 -F691L mutations at progression. Two patterns of resistance to gilteritinib were identified based on NGS data: 1) “clone swapping” in which the original FLT3 mutation is lost with the concurrent acquisition of mutations in other genes and 2) “clonal evolution” in which baseline mutations in FLT3 and other genes persist but additional mutations are acquired, causing subclonal expansion (Figure 1).Conclusions: Most patients responding to gilteritinib maintain a stable marrow FLT3 mutant allele burden, indicating terminal differentiation of leukemic blasts. Treatment-emergent mutational activation of signal transduction underlies the majority of cases of gilteritinib resistance, but only a small number-all F691L-are new FLT3 mutations. Cytogenetic clonal evolution during gilteritinib occurs commonly but is not always associated with resistance. Mechanisms of gilteritinib resistance include the acquisition of new driver mutations and selection for FLT3 -WT subclones. Dual targeting of NRAS signaling and FLT3 may prove useful clinically. [Display omitted] DisclosuresMorrissette:Novartis: Consultancy. Shah:Daiichi-Sankyo: Research Funding; ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding. Smith:Astellas Pharma: Research Funding; Plexxikon Inc.: Research Funding. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding. Perl:Novartis: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Arog Pharmaceuticals: Consultancy; Pfizer: Other: Advisory Board; Asana Biosciences: Other: Scientific advisory board; Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Astellas: Consultancy.

Treatment Patterns and Healthcare Resource Utilization in Patients with FLT3-Mut and FLT3-Wt Acute Myeloid Leukemia: A Multi-Country Medical Chart Study

Introduction: FLT3 is a frequently mutated gene in acute myeloid leukemia (AML) with two main types of mutations: internal tandem duplication (ITD) and point mutations in the tyrosine kinase domain (TKD). FLT-mut AML is associated with poor prognosis. With the development of new therapies in AML, especially those targeting FLT3-mut, there is a need to understand the current treatment (tx) patterns and healthcare resource utilization (HRU) among AML patients. The current study used real-world data from medical records to evaluate tx patterns and HRU among adult AML patients.Methods: Hematologists and oncologists were recruited from 10 countries (US, Canada, UK, France, Germany, Italy, Spain, Netherlands, Japan, and South Korea) from an established physician panel. Eligible patients were randomly selected by physicians and were categorized into 6 cohorts: 1) newly diagnosed (ND) FLT3-mut patients <65, 2) ND FLT3-mut patients ≥65, 3) ND FLT3-wild type (FLT3-wt) patients <65, 4) ND FLT3-wt patients ≥65, 5) relapsed/refractory (R/R) FLT3-mut patients, and 6) R/R FLT3-wt patients. The index date was defined as the date of initiation of first AML therapy for ND patients, and the date of R/R classification for R/R patients. FLT3-mut was based on the genetic test closest to the index date. FLT3-mut and FLT3-wt patients may harbor mutations in other genes. Baseline characteristics, tx patterns, and AML-related HRU were collected and described for each cohort.Results: The study included 1,027 AML patients-183 FLT3-mut and 186 FLT3-wt ND patients <65, 136 and 159 ND patients ≥65, and 181 and 182 R/R patients. Mean age was 48.2 and 72.3 for ND patients <65 and ≥65; among R/R patients, mean age of FLT3-mut patients was younger than FLT3-wt patients (53.2 vs. 56.8). Among FLT3-mut patients, 70.0% had ITD and 42.4% had TKD mutations. The study identified substantial heterogeneity in tx pattern for AML. Among ND patients <65, the most common initial tx was standard-to-intermediate dose cytarabine (SDAC)-based therapies (43.2 and 55.9% for FLT3-mut and FLT3-wt), followed by hypomethylating agent (HMA)-based therapies (13.7 and 11.8%). Among ND patients ≥65, the most common initial tx were HMA-based therapies (36.0 and 47.2%), followed by SDAC-based therapies (30.1 and 30.8%). Among R/R patients, the most common initial tx after R/R was BSC only (39.8 and 24.7%), followed by SDAC-based therapies (12.7 and 19.2%), HMA-based therapies (9.4 and 16.5%), and low dose cytarabine-based therapies (9.4 and 15.4%). About 20-60% ND patients and 40% R/R patients received non-guideline recommended tx. Among ND patients, tx for FLT3-mut patients tended to be more aggressive than for FLT3-wt patients. For patients <65, FLT3-mut patients used more high-dose cytarabine-based therapies than FLT3-wt patients (13.7 vs 9.7%); for patients ≥65, fewer FLT3-mut patients used HMA-based therapies than FLT3-wt patients (36.0 vs 47.2%). The proportion of patients who received stem cell transplant was higher in FLT3-mut vs. FLT3-wt patients (ND<65: 29.2 vs. 24.3%; ND≥65: 13.6 vs. 8.5%; R/R: 23.6 vs. 18.1%). In addition, this study demonstrated that AML patients had extensive HRU. The average rate of hospitalization across all cohorts during the event-free period (i.e., the period free of R/R for ND cohorts or before next R/R for R/R cohorts) was 0.27 hosp/mo (5.4 d/mo), while the rate during the post-event period was 0.52 hosp/mo (6.5 d/mo). The average number of days of intensive care unit stays was 0.28 d/mo for the event-free period and 0.50 d/mo for the post-event period. For emergency department visits, the average rate was 0.23 visits/mo during the event-free period and 0.54 visits/mo during the post-event period. During both event-free and post-event periods, patients across all cohorts also experienced frequent outpatient visits, blood transfusions, and received extensive tx for infections.Conclusions: Using real-world data of AML patients in multiple countries, this study reveals a considerable amount of heterogeneity of tx pattern, including many tx not consistent with tx guidelines. FLT3-mut patients tended to receive more aggressive tx, consistent with fact that the mutation confers a poor prognosis. It also demonstrates extensive HRU among these patients, particularly among R/R cohorts. The study provides timely evidence to understand the current tx landscape and to highlight the substantial unmet needs among AML patients. DisclosuresGriffin:Novartis: Consultancy, Research Funding; Astellas Pharma Global Development: Consultancy. Yang:Analysis Group Inc.: Employment; Novartis Pharmaceutical Corporation: Other: Author is an employee of Analysis Group, which received consulting fees from Novartis for this study. Song:Analysis Group: Employment. Kinrich:Analysis Group: Employment. Bui:Astellas Pharma Global Development: Employment.

CG '806, a Novel Pan-FLT3/BTK Multi-Kinase Inhibitor, Induces Cell Cycle Arrest, Apoptosis or Autophagy in AML Cells Depending on FLT3 Mutation Status

Gain-of-function mutations of the fms-like tyrosine kinase 3 (FLT3) play a pivotal role in hematopoietic malignancies (Gilliland et al., 2002). Therefore, FLT3 kinase inhibitors are important component in the treatment of acute myeloid leukemia (AML). However, relapse or resistance to these inhibitors are frequent because of secondary acquired mutations in FLT3. Recently, we reported on a novel small molecular multi-kinase inhibitor, CG'806, which showed promising effects in AML harboring FLT3-internal-tandem-deletion (ITD) mutations, tyrosine kinase domain (TKD) mutations, or both by inhibiting FLT3/Bruton's tyrosine kinase (BTK)/aurora kinase (AuroK) activation. We observed an impressive inhibition of leukemic cell proliferation (i.e., IC50s at sub-nanomolar or low nanomolar levels, Zhang, et al., AACR Hematological Malignancies, 2017).To further characterize the mechanisms underlying this anti-leukemia effect, we investigated the impact of CG'806 on cell cycle progression. Measuring BrdU incorporation by flow cytometry, CG'806 triggered profound G1 cell cycle arrest in FLT3-ITD-mutated cells. Interestingly, CG'806 triggered G2/M phase arrest in FLT3 wild type (WT) cells. Immunoblotting demonstrated that the G1 arrest was mediated by downregulation of signaling associated with p-FLT3-ITD, the p-AKT/p-mTOR/cyclin D1/p-Rb axis, and that of cyclin B1/A2, cdk1/cdc2, and cdk4/6 in FLT3-ITD-mutated cells. This was not observed in FLT3 WT cells. In addition, c-Myc, a primary regulator of the G1/S transition, was downregulated in FLT3-mutated but not in WT cells treated with CG'806. In support of this finding, knockdown of c-Myc with siRNA increased the G1-arrested population in FLT3-mutated cells, suggesting a critical role of c-Myc in the CG'806-induced G1 arrest. However, only suppression of p-BTK and p-AuroK but not p-FLT3, and no modulation of G1 arrest-related proteins was observed in FLT3 WT cells.Interestingly, we observed autophagy induction in FLT3 WT cells but induction of apoptosis in FLT3 mutated cells after exposure to low nanomolar CG'806 for 24 hours as evidenced by modulated LC3-II and cleaved-caspase 3 levels. Inhibiting autophagy with 3-methyladenine (3-MA) partially reversed CG'806-induced G2/M arrest in FLT3 WT cells, suggesting that autophagy induction may also be involved in G2/M arrest besides suppression of AuroK and BTK in FLT3 WT cells.Next, we investigated if nucleoside analogues and intercalating agents enhance activity of CG'806, and observed that combination of CG'806 with cytarabine or idarubicin profoundly enhanced pro-apoptotic effects.Conclusions: CG'806 exerts profound suppression of cell proliferation by arresting cell cycle progression at G1 phase in FLT3-mutant AML cells which is associated with inhibition of mutant FLT3 and downstream p-AKT/p-mTOR/cyclin D1/p-Rb axis. CG'806 exerts a G2/M arrest in FLT3 WT cells associated with inhibition of aurora and BTK kinases and induction of autophagy. CG'806 sensitized AML to standard chemotherapeutic agents-mediated cytotoxicity. Taken together, these data support the development of CG'806 for AML patients with FLT3-ITD, FLT3-ITD with additional TKD mutations, as well as FLT3-WT. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Concomitant Use of Midostaurin with Strong CYP3A4 Inhibitors: An Analysis from the Ratify Trial

Introduction: Midostaurin is an oral multikinase inhibitor with activity in FMS-like tyrosine kinase 3 (FLT3 )-mutated acute myeloid leukemia (AML), including internal tandem duplications and tyrosine kinase domain mutations. The phase 3, double-blind, placebo-controlled, randomized RATIFY/CALGB (Alliance) 10603 trial investigated the effect of midostaurin in combination with standard chemotherapy and as single-agent maintenance therapy on outcomes in patients with newly diagnosed FLT3 -mutated AML. When added to standard chemotherapy, midostaurin improved both event-free survival (EFS) and overall survival (OS) (Stone et al, N Engl J Med. 2017 Jun 23. [Epub ahead of print]). Midostaurin is metabolized into 2 metabolites (CGP62221 and CGP52421) by cytochrome P450 3A4 (CYP3A4). We evaluated the effects of strong CYP3A4 inhibitors on midostaurin exposure, activity, and safety based on clinical data from the RATIFY trial.Methods: Patients received placebo or midostaurin 50 mg twice daily on days 8 to 21 of each 28-day induction or consolidation cycle, followed by 50 mg twice daily as a single agent on days 1 to 28 during the 12-cycle maintenance phase. Concomitant medication use was recorded for patients throughout all 3 treatment phases (induction, consolidation, and maintenance). Plasma levels of midostaurin and its metabolites were measured in a subset of 188 patients. Data on grade ≥3 adverse events (AEs) were collected at all sites; all grade 1/2 AE data were collected by sites outside the United States while only 13 prespecified grade 1/2 AEs were recorded at North American sites. The effect of CYP3A4 on efficacy was assessed using multiple endpoints including complete response, EFS, and OS.Results: Concomitant medication use was balanced between the midostaurin and placebo groups. Strong CYP3A4 inhibitors concurrently administered most frequently were the antifungal medications posaconazole and voriconazole. Strong CYP3A4 inhibitors were used in 60.8%, 45.6%, and 10.8% of patients during induction, consolidation, and maintenance, respectively.A 1.44-fold increase in midostaurin exposure (Cmin) was observed in patients receiving coadministration of strong CYP3A4 inhibitors compared with patients not receiving strong CYP3A4 inhibitors (number of patients: n=55 vs n=112, respectively). No relevant effect was observed with CGP62221 and CGP52421. Because the protocol did not include any recommendations for dose modifications when strong CYP3A4 inhibitors were administered, the median relative dose intensity of midostaurin was >94% of the intended dose in both treatment arms. There was no notable increase in midostaurin-related AEs in patients receiving strong CYP3A4 inhibitors. Exposure-safety analyses showed a slightly shorter time to occurrence of grade 3 or 4 clinically notable AEs with increasing exposure, with an overall acceptable safety profile for midostaurin. Exposure-response analyses indicated that a higher dose intensity was associated with a larger benefit to patients, namely through complete response, EFS and OS. This observation was generally not affected by demographic covariates or presence/absence of CYP3A4 perpetrators during the cycle where the PK sample was used for the analysis.Conclusions: Overall, co-administration of midostaurin with strong CYP3A4 inhibitors, without midostaurin dose adjustment, led to a balanced safety and efficacy profile. Because strong CYP3A4 inhibitors can increase the plasma concentration of midostaurin, caution is required when concomitantly prescribing these with midostaurin. Alternative medicinal products that do not strongly inhibit CYP3A4 activity should be considered. In situations in which satisfactory therapeutic alternatives do not exist, patients should be closely monitored for midostaurin-related toxicity.Support: U10CA180821, U10CA180882, U24CA196171; ClinicalTrials.gov identifier, NCT00651261. DisclosuresOuatas:Novartis: Employment. Duval:Novartis: Employment. Sinclair:Novartis: Employment. Berkowitz:Novartis: Employment.