Trivikram Rajkhowa

Phase 2 study of azacytidine plus sorafenib in patients with acute myeloid leukemia and FLT-3 internal tandem duplication mutation

Patients received 5-azacytidine (AZA) 75 mg/m(2) intravenously daily for 7 days and sorafenib 400 mg orally twice daily continuously; cycles were repeated at ~1-month intervals. Forty-three acute myeloid leukemia (AML) patients with a median age of 64 years (range, 24-87 years) were enrolled; 37 were evaluable for response. FMS-like tyrosine kinase-3 (FLT3)-internal tandem duplication (ITD) mutation was detected in 40 (93%) patients, with a median allelic ratio of 0.32 (range, 0.009-0.93). They had received a median of 2 prior treatment regimens (range, 0-7); 9 had failed prior therapy with a FLT3 kinase inhibitor. The response rate was 46%, including 10 (27%) complete response with incomplete count recovery (CRi), 6 (16%) complete responses (CR), and 1 (3%) partial response. The median time to achieve CR/CRi was 2 cycles (range, 1-4), and the median duration of CR/CRi was 2.3 months (range, 1-14.3 months). Sixty-four percent of patients achieved adequate (defined as >85%) FLT3 inhibition during their first cycle of therapy. The degree of FLT3 inhibition correlated with plasma sorafenib concentrations. FLT3 ligand levels did not rise to levels seen in prior studies of patients receiving cytotoxic chemotherapy. The combination of AZA and sorafenib is effective for patients with relapsed AML and FLT-3-ITD. This trial was registered at clinicaltrials.gov as #NCT01254890.

FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML

We examined 6 different FMS-like tyrosine kinase-3 (FLT3) inhibitors (lestaurtinib, midostaurin, AC220, KW-2449, sorafenib, and sunitinib) for potency against mutant and wild-type FLT3, as well as for cytotoxic effect against a series of primary blast samples obtained from patients with acute myeloid leukemia (AML) harboring internal tandem duplication (FLT3/ITD) mutations. We found that inhibition of FLT3 autophosphorylation in a FLT3/ITD specimen does not always induce cell death, suggesting that some FLT3/ITD AML may not be addicted to FLT3 signaling. Relapsed samples and samples with a high mutant allelic burden were more likely to be responsive to cytotoxicity from FLT3 inhibition compared with the samples obtained at diagnosis or those with a low mutant allelic burden. These FLT3 inhibitors varied to a considerable degree in their selectivity for FLT3, and this selectivity influenced the cytotoxic effect. These results have important implications for the potential therapeutic use of FLT3 inhibitors in that patients with newly diagnosed FLT3-mutant AML might be less likely to respond clinically to highly selective FLT3 inhibition.

Crenolanib is a potent inhibitor of FLT3 with activity against resistance-conferring point mutants.

Mutations of the type III receptor tyrosine kinase FLT3 occur in approximately 30% of acute myeloid leukemia patients and lead to constitutive activation. This has made FLT3-activating mutations an attractive drug target because they are probable driver mutations of this disease. As more potent FLT3 inhibitors are developed, a predictable development of resistance-conferring point mutations, commonly at residue D835, has been observed. Crenolanib is a highly selective and potent FLT3 tyrosine kinase inhibitor (TKI) with activity against the internal tandem duplication (FLT3/ITD) mutants and the FLT3/D835 point mutants. We tested crenolanib against a panel of D835 mutant cell lines and primary patient blasts and observed superior cytotoxic effects when compared with other available FLT3 TKIs such as quizartinib and sorafenib. Another potential advantage of crenolanib is its reduced inhibition of c-Kit compared with quizartinib. In progenitor cell assays, crenolanib was less disruptive of erythroid colony growth, which may result in relatively less myelosuppression than quizartinib. Finally, correlative data from an ongoing clinical trial demonstrate that acute myeloid leukemia patients can achieve sufficient levels of crenolanib to inhibit both FLT3/ITD and resistance-conferring FLT3/D835 mutants in vivo. Crenolanib is thus an important next-generation FLT3 TKI.

Terminal myeloid differentiation in vivo is induced by FLT3 inhibition in FLT3/ITD AML

A hallmark of cancer is the disruption of differentiation within tumor cells. Internal tandem duplication mutations of the FLT3 kinase (FLT3/ITD) occur commonly in acute myeloid leukemia (AML) and are associated with poor survival, leading to efforts to develop FLT3 kinase inhibitors. However, FLT3 inhibitors have thus far met with limited success, inducing only a clearance of peripheral blasts with minimal BM responses. Quizartinib is a novel potent and selective FLT3 inhibitor currently being studied in clinical trials. In 13 of 14 FLT3/ITD AML patients with normal karyotype treated with quizartinib, we observed terminal myeloid differentiation of BM blasts in association with a clinical differentiation syndrome. The single patient whose blasts failed to differentiate had a preexisting C/EBPα mutation and another developed a C/EBPα mutation at disease progression, suggesting a mechanism of resistance to FLT3 inhibition. In vitro, in primary blasts cocultured with human BM stroma, FLT3 inhibition with quizartinib induced cell-cycle arrest and differentiation rather than apoptosis. The present study is the first description of terminal differentiation of cancer cells in patients treated with a tyrosine kinase inhibitor. These data highlight the importance of the differentiation block in the patho-genesis of AML.

Phase I/II trial of the combination of midostaurin (PKC412) and 5-azacytidine for patients with acute myeloid leukemia and myelodysplastic syndrome

We investigated the combination of midostaurin and azacitidine (AZA) in patients with acute myeloid leukemia (AML) and high risk myelodysplastic syndrome (MDS). Patients received AZA 75 mg m−2 on days 1–7 and midostaurin 25 mg bid (in cohort 1 of phase I) or 50 mg bid (in cohort 2 of Phase I and in Phase II) orally on day 8–21 during the first cycle and continuously thereafter. Fourteen patients were enrolled in the phase I and 40 in the phase II. Overall response rate was 26%. The median remission duration (RD) was 20 weeks and was significantly longer in patients with FLT3 mutations not previously exposed to other FLT3 inhibitors (P = 0.05) and in patients not previously transplanted (P = 0.01). Thirty‐two (59%) patients have died, all of complications related to disease progression. G3‐4 nonhematological toxicity was reported in 38 (70%) patients, most frequently infections (56%), ejection fraction reduction (11%), and diarrhea or nausea/vomiting (9% each). The combination of midostaurin and AZA is an effective and safe regimen in patients with AML and high‐risk MDS. Patients with FLT3 mutations but not previously exposed to other FLT3 inhibitors and patients not previously transplanted derived the greatest benefit. Further studies with this combination are warranted. Am. J. Hematol. 90:276–281, 2015.

A potential therapeutic target for FLT3-ITD AML: PIM1 Kinase

Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) mutation have a poor prognosis, and FLT3 inhibitors are now under clinical investigation. PIM1, a serine/threonine kinase, is up-regulated in FLT3-ITD AML and may be involved in FLT3-mediated leukemogenesis. We employed a PIM1 inhibitor, AR00459339 (Array Biopharma Inc.), to investigate the effect of PIM1 inhibition in FLT3-mutant AML. Like FLT3 inhibitors, AR00459339 was preferentially cytotoxic to FLT3-ITD cells, as demonstrated in the MV4-11, Molm-14, and TF/ITD cell lines, as well as 12 FLT3-ITD primary samples. Unlike FLT3 inhibitors, AR00459339 did not suppress phosphorylation of FLT3, but did promote the de-phosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339 with a FLT3 inhibitor resulted in additive to mildly synergistic cytotoxic effects. AR00459339 was cytotoxic to FLT3-ITD samples from patients with secondary resistance to FLT3 inhibitors, suggesting a novel benefit to combining these agents. We conclude that PIM1 appears to be closely associated with FLT3 signaling, and that inhibition of PIM1 may hold therapeutic promise, either as monotherapy, or by overcoming resistance to FLT3 inhibitors.

Pre-clinical studies of gilteritinib, a next-generation FLT3 inhibitor.

To the editor: FLT3-activating mutations are one of the most frequent genetic aberrations in acute myeloid leukemia (AML).[1][1] Internal tandem duplication (FLT3-ITD) mutations are associated with the worst prognosis, whereas tyrosine kinase domain (FLT3/TKD) mutations have an uncertain prognostic

Expression of putative targets of immunotherapy in acute myeloid leukemia and healthy tissues

The ability to target myeloid malignancies using immunotherapy through means other than allogeneic transplantation depends on the capability to target leukemic clones while sparing normal tissues. It is now possible to generate clinical grade ex-vivo expanded T cells specific for leukemia-associated antigens (LAAs) for use in adoptive cell therapy.1 Although a variety of putative LAAs in acute myeloid leukemia (AML) have been identified for use as potential targets for immunotherapy2, 3, 4, 5, 6, 7, 8 and consensus panels have attempted to prioritize generic cancer antigens,9 a comprehensive evidence-based list of AML antigen targets has not yet been established. As a first step toward this goal, we therefore analyzed, using quantitative real-time PCR, the gene expression of 65 potential LAAs (Supplementary Table S1) in de-identified, clinically annotated samples from 48 newly diagnosed untreated AML patients that were collected under institutional review board-approved protocols from three NCCN cancer centers.

Abstract 3660: Crenolanib: A next generation FLT3 inhibitor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Every year in the US there are approximately 12,000 new cases of acute myeloid leukemia (AML). Amongst these patients, 25% have internal tandem duplications (ITDs) of the type III receptor tyrosine kinase, FLT3, causing constitutive activation of the receptor. This mutation has been associated with a poor survival rate, thus making the FLT3/ITD an important target in the treatment of AML. Treatment with small molecule tyrosine kinase inhibitors (TKIs) has shown high response rates in AML patients.1 However, this response is often temporary due to the development of resistance-conferring point mutations.2 Furthermore, QTc prolongation has proven to be a dose-limiting toxicity with some FLT3 TKIs.1 Crenolanib is a TKI with activity against PDGFR and FLT3 and is currently being tested in trials involving patients with a variety of solid tumors. We have determined that crenolanib has potent activity in vitro against FLT3. Crenolanib is cytotoxic to the FLT3/ITD-expressing leukemia cell lines Molm14 and MV411, with an IC50 of 7 nM and 8 nM, respectively. In immunoblots, crenolanib inhibited phosphorylation of both the wild type FLT3 receptor (in SEMK2 cells) and the FLT3/ITD receptor (in Molm14 cells) in culture medium with an IC50 of 1-3 nM. Importantly, the IC50 of crenolanib against the D835Y mutated form of FLT3 was 8.8 nM in culture medium. Using primary AML patient samples, we determined that crenolanib was cytotoxic to FLT3/ITD-expressing samples in a manner comparable to agents such as sorafenib and AC220. Furthermore, crenolanib had cytotoxic activity against primary samples that were obtained from patients who had developed D835 resistance mutations while receiving FLT3 TKIs. In vitro, the IC50 of crenolanib for inhibition of FLT3/ITD in plasma was found to be 34 nM, indicating a relatively low degree of plasma protein binding. From pharmacokinetic studies of crenolanib in solid tumor patients, steady state trough plasma levels of roughly 500 nM were found to be safe and tolerable, suggesting that crenolanib could potentially inhibit the target in vivo as effectively as AC220. Unlike AC220, crenolanib has no significant activity against c-KIT, which may be an advantage in that myelosuppression can be avoided. Furthermore, there was no evidence of QTc prolongation in patients treated with crenolanib. In summary, crenolanib offers a number of advantages over other FLT3 TKIs. Clinical trials of crenolanib in AML patients with FLT3 activating mutations are planned. References: 1) Cortes et al. (2011) A Phase II Open-Label, AC220 Monotherapy Efficacy Study in Patients with Refractory/Relapsed Flt3-Itd Positive Acute Myeloid Leukemia: Updated Interim Results. American Society of Hematology Abstract no. 2576. 2) Smith et al (2011) Validation of FLT3-ITD As a Therapeutic Target in Human Acute Myeloid Leukemia. American Society of Hematology Abstract no. 937. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3660. doi:1538-7445.AM2012-3660