Divergent cytotoxic effects of PKC412 in combination with conventional antileukemic agents in FLT3 mutation-positive versus -negative leukemia cell lines.

FMS-like tyrosine kinase-3 (FLT3) is a new therapeutic target for acute myelocytic leukemia (AML), because FLT3 mutations are the most common genetic alterations in AML and are directly related to leukemogenesis. We studied cytotoxic interactions of a FLT3 inhibitor, PKC412, with eight conventional antileukemic agents (cytarabine, doxorubicin, idarubicin, mitoxantrone, etoposide, 4-hydroperoxy-cyclophosphamide, methotrexate and vincristine) using three leukemia cell lines carrying FLT3 mutations (MOLM13, MOLM14 and MV4-11) and five leukemia cell lines without FLT3 mutations (KOPB-26, THP-1, BALL-1, KG-1 and U937). PKC412 showed synergistic effects with all agents studied except methotrexate for FLT3-mutated cell lines in isobologram analysis. In contrast, PKC412 was rather antagonistic to most drugs, except for 4-hydroperoxy-cyclophosphamide and vincristine, in leukemia cell lines without FLT3 mutations. Cell-cycle analysis revealed that PKC412 induced G1 arrest in leukemia cell lines carrying FLT3 mutations, whereas it arrested cells in G2/M phase in the absence of FLT3 mutations, which may underlie the divergent cytotoxic interactions. These results suggest that the simultaneous administration of PKC412 and other agents except methotrexate is clinically effective against FLT3 mutation-positive leukemias, whereas it would be of little benefit for FLT3 mutation-negative leukemias. Our findings may be of help for the design of PKC412-based combination chemotherapy.     

The FLT3 inhibitor PKC412 exerts differential cell cycle effects on leukemic cells depending on the presence of FLT3 mutations

PKC412 is a staurosporine derivative that inhibits several protein kinases including FLT3, and is highly anticipated as a novel therapeutic agent for acute myeloblastic leukemia (AML) carrying FLT3 mutations. In this study, we show that PKC412 exerts differential cell cycle effects on AML cells depending on the presence of FLT3 mutations. PKC412 elicits massive apoptosis without markedly affecting cell cycle patterns in AML cell lines with FLT3 mutations (MV4-11 and MOLM13), whereas it induces G2 arrest but not apoptosis in AML cell lines without FLT3 mutations (THP-1 and U937). In MV4-11 and MOLM13 cells, PKC412 inactivates Myt-1 and activates CDC25c, leading to the activation of CDC2. Activated CDC2 phosphorylates Bad at serine-128 and facilitates its translocation to the mitochondria, where Bad triggers apoptosis. In contrast, PKC412 inactivates CDC2 by inducing serine-216 phosphorylation and subsequent cytoplasmic sequestration of CDC25c in THP-1 and U937 cells. As a result, cells are arrested in the G2 phase of the cell cycle, but do not undergo apoptosis because Bad is not activated. The FLT3 mutation-dependent differential cell cycle effect of PKC412 is considered an important factor when PKC412 is combined with cell cycle-specific anticancer drugs in the treatment of cancer and leukemia.     

Novel AXL-targeted agents overcome FLT3 inhibitor resistance in FLT3-ITD+ acute myeloid leukemia cells

AXL receptor tyrosine kinase (AXL) upregulation mediates drug resistance in several types of human cancer and has become a therapeutic target worthy of exploration. The present study investigated AXL antigen expression and the effects of novel AXL-targeted agents in acute myeloid leukemia (AML) cells. AXL antigen expression in drug-sensitive and drug-resistant human AML cell lines, and AML blast cells from 57 patients with different clinical characteristics, was analyzed by flow cytometry and compared. Furthermore, the effects of the novel AXL antibody DAXL-88, antibody-drug conjugate DAXL-88-monomethyl auristatin E (MMAE), AXL small molecule inhibitor R428 and their combination with FMS-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) in AML cells were analyzed by Cell Counting Kit-8 assay, flow cytometry and western blotting. The present study revealed that AXL antigen expression was upregulated in FLT3-internal tandem duplication (ITD)/tyrosine kinase domain mutation-positive (TKD)⁺ AML blast cells compared with FLT3-ITD/TKD⁻ AML cells. Additionally, AXL antigen expression was markedly upregulated in the AC220-resistant FLT3-ITD⁺ MV4-11 cell line (MV4-11/AC220) and in FLT3 inhibitor-resistant blast cells from a patient with FLT3-ITD⁺ AML compared with parental sensitive cells. The AXL-targeted agents DAXL-88, DAXL-88-MMAE and R428 exhibited dose-dependent cytotoxic effects on FLT3-mutant AML cell lines (THP-1, MV4-11 and MV4-11/AC220) and blast cells from patients with FLT3-ITD⁺ AML. Combinations of AXL-targeted agents with AC220 exerted synergistic cytotoxic effects and induced apoptosis in MV4-11/AC220 cells and FLT3 inhibitor-resistant blast cells. The antileukemic effect of DAXL-88 and DAXL-88-MMAE may rely on their ability to block AXL, FLT3 and their downstream signaling pathways. The present study demonstrated the association between AXL antigen expression upregulation and drug resistance in FLT3-ITD⁺ AML, and proposed a method for overcoming FLT3 inhibitor resistance of FLT3-ITD⁺ AML using novel AXL-targeted agents.     

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.     

Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412

Constitutively activating FLT3 receptor mutations have been found in 35% of patients with acute myeloblastic leukemia (AML). Here we report the identification of a small molecule FLT3 tyrosine kinase inhibitor PKC412, which selectively induced G1 arrest and apoptosis of Ba/F3 cell lines expressing mutant FLT3 (IC(50) < 10 nM) by directly inhibiting the tyrosine kinase. Ba/F3-FLT3 cell lines made resistant to PKC412 demonstrated overexpression of mutant FLT3, confirming that FLT3 is the target of this drug. Finally, progressive leukemia was prevented in PKC412-treated Balb/c mice transplanted with marrow transduced with a FLT3-ITD-expressing retrovirus. PKC412 is a potent inhibitor of mutant FLT3 and is a candidate for testing as an antileukemia agent in AML patients with mutant FLT3 receptors.     

Cotreatment with 17-allylamino-demethoxygeldanamycin and FLT-3 kinase inhibitor PKC412 is highly effective against human acute myelogenous leukemia cells with mutant FLT-3

Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G(1) phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.     

The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

About 25% of patients with acute myeloid leukemia (AML) harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations and their prognosis remains poor. Gilteritinib is a FLT3 inhibitor approved by the US FDA for use in adult FLT3-mutated relapsed or refractory AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Here we show that gilteritinib and CUDC-907, a dual inhibitor of PI3K and histone deacetylases, synergistically induce apoptosis in FLT3-ITD AML cell lines and primary patient samples and have striking in vivo efficacy. Upregulation of FLT3 and activation of ERK are mechanisms of resistance to gilteritinib, while activation of JAK2/STAT5 is a mechanism of resistance to CUDC-907. Gilteritinib and CUDC-907 reciprocally overcome these mechanisms of resistance. In addition, the combined treatment results in cooperative downregulation of cellular metabolites and persisting antileukemic effects. CUDC-907 plus gilteritinib shows synergistic antileukemic activity against FLT3-ITD AML in vitro and in vivo, demonstrating strong translational therapeutic potential.Subject terms: Acute myeloid leukaemia, Drug development     

Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells

FLT3 mutation and overexpression in most acute myeloid leukaemia (AML) patients make this tyrosine kinase receptor an attractive therapeutic target. FLT3 kinase inhibitors are actually in clinical trials, thus it is critical to develop a reproducible and standardized method for screening of FLT3 activation and for monitoring its inhibition in response to drug in AML patients. We developed a flow cytometry method to analyse phosphorylated FLT3 (P-FLT3) in samples with <10(5) cells. The method was first validated in FLT3 wild-type (HL60/WT) and mutant (MV4-11/ITD(+)) as well as FLT3 negative (K562) cell lines. The method also proved to be reproducible in AML patient samples. Analysis was performed after exposure to drugs (CEP-701 and SU11657), in vitro and in vivo. In response to increasing drug concentrations, there was a linear reduction in P-FLT3. Intracellular flow cytometry analysis correlated with Western blot and XTT assays; flow cytometry data also correlated with FLT3 mutational status. The results highlight a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry which enables an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.     

Association of the EGF-TM7 receptor CD97 expression with FLT3-ITD in acute myeloid leukemia

Internal tandem duplications within the juxtamembrane region of the FMS-like tyrosine kinase receptor FLT3 (FLT3-ITD) represents one of the most common mutations in patients with acute myeloid leukemia (AML) which results in constitutive aberrant activation, increased proliferation of leukemic progenitors and is associated with an aggressive clinical phenotype. The expression of CD97, an EGF-TM7 receptor, has been linked to invasive behavior in thyroid and colorectal cancer. Here, we have investigated the association of CD97 with FLT3-ITD and its functional consequences in AML.Higher CD97 expression levels have been detected in 208 out of 385 primary AML samples. This was accompanied by a significantly increased bone marrow blast count as well as by mutations in the FLT3 gene. FLT3-ITD expressing cell lines as MV4-11 and MOLM-13 revealed significantly higher CD97 levels than FLT3 wildtype EOL-1, OCI-AML3 and HL-60 cells which were clearly decreased by the tyrosine kinase inhibitors PKC412 and SU5614. CD97 knock down by short hairpin RNA in MV4-11 cells resulted in inhibited trans-well migration towards fetal calf serum (FCS) and lysophosphatidic acid (LPA) being at least in part Rho-A dependent. Moreover, knock down of CD97 led to an altered mechanical phenotype, reduced adhesion to a stromal layer and lower wildtype FLT3 expression.Our results, thus, constitute the first evidence for the functional relevance of CD97 expression in FLT3-ITD AML cells rendering it a potential new theragnostic target.     

Acquisition of FLT3 or N-ras mutations is frequently associated with progression of myelodysplastic syndrome to acute myeloid leukemia.

The role of internal tandem duplication of fms-like tyrosine kinase 3 (FLT3/ITD), mutations at tyrosine kinase domain (FLT3/TKD) and N-ras mutations in the transformation of myelodysplastic syndrome (MDS) to AML was investigated in 82 MDS patients who later progressed to AML; 70 of them had paired marrow samples at diagnosis of MDS and AML available for comparative analysis. Five of the 82 patients had FLT3/ITD at presentation. Of the 70 paired samples, seven patients acquired FLT3/ITD during AML evolution. The incidence of FLT3/ITD at diagnosis of MDS was significantly lower than that at AML transformation (3/70 vs 10/70, P<0.001). FLT3/ITD(+) patients progressed to AML more rapidly than FLT3/ITD(-) patients (2.5+/-0.5 vs 11.9+/-1.5 months, P=0.114). FLT3/ITD(+) patients had a significantly shorter survival than FLT3/ITD(-) patients (5.6+/-1.3 vs 18.0+/-1.7 months, P=0.0008). After AML transformation, FLT3/ITD was also associated with an adverse prognosis. One patient had FLT3/TKD mutation (D835Y) at both MDS and AML stages. Additional three acquired FLT3/TKD (one each with D835 H, D835F and I836S) at AML transformation. Five of the 70 matched samples had N-ras mutation at diagnosis of MDS compared to 15 at AML transformation (P<0.001), one lost and 11 gained N-ras mutations at AML progression. Coexistence of FLT3/TKD and N-ras mutations was found in two AML samples. N-ras mutations had no prognostic impact either at the MDS or AML stage. Our results show that one-third of MDS patients acquire activating mutations of FLT3 or N-ras gene during AML evolution and FLT3/ITD predicts a poor outcome in MDS.     

Superior activity of the combination of histone deacetylase inhibitor LAQ824 and the FLT-3 kinase inhibitor PKC412 against human acute myelogenous leukemia cells with mutant FLT-3.

PURPOSE: Mutant FLT-3 receptor tyrosine kinase is a client protein of the molecular chaperone heat shock protein 90 and is commonly present and contributes to the leukemia phenotype in acute myelogenous leukemia (AML). LAQ824, a cinnamyl hydroxamate histone deacetylase inhibitor, is known to induce acetylation and inhibition of heat shock protein 90. Here, we determined the effects of LAQ824 and/or PKC412 (a FLT-3 kinase inhibitor) on the levels of mutant FLT-3 and its downstream signaling, as well as growth arrest and cell-death of cultured and primary human AML cells. EXPERIMENTAL DESIGN: The effect of LAQ824 and/or PKC412 treatment was determined on the levels of FLT-3 and phosphorylated (p)-FLT-3, on downstream pro-growth and pro-survival effectors, e.g., p-STAT5, p-AKT, and p-extracellular signal-regulated kinase (ERK) 1/2, and on the cell cycle status and apoptosis in the cultured MV4-11 and primary AML cells with mutant FLT-3. RESULTS: Treatment with LAQ824 promoted proteasomal degradation and attenuation of the levels of FLT-3 and p-FLT-3, associated with cell cycle G(1)-phase accumulation and apoptosis of MV4-11 cells. This was accompanied by attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels. STAT-5 DNA-binding activity and the levels of c-Myc and oncostatin M were also down-regulated. Cotreatment with LAQ824 and PKC412 synergistically induced apoptosis of MV4-11 cells and induced more apoptosis of the primary AML cells expressing mutant FLT-3. This was also associated with more attenuation of p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5. CONCLUSIONS: The combination of LAQ824 and PKC412 is highly active against human AML cells with mutant FLT-3, which merits in vivo studies of the combination against human AML.     

PKC 412 small-molecule tyrosine kinase inhibitor: single-compound therapy for pancreatic cancer

BACKGROUND: PKC412 is a kinase inhibitor that blocks protein kinase C (PKC), vascular endothelial growth factor receptors, platelet-derived growth factor receptor FLT3, and other class III receptor tyrosine kinases. The enthusiasm for this compound is based on its inhibitory effect even in the case of FLT3 mutations. The aim of this study was to analyze the role of FLT3 in pancreatic cancer and to study the biological activity of combined inhibition of neovascularization and mitogenesis in this disease. METHODS: FLT3 expression was analyzed in 18 pancreatic cancer specimens by real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Sixteen pancreatic cancer cell lines were screened for ITD and D835 point mutations of the FLT3 gene. MTT assays and anchorage-independent growth assays were used to study cell growth. Flow cytometry was used for cell cycle analysis and apoptosis quantification. In vivo AsPC-1 and HPAF-II cells were used for orthotopic tumor modeling. Immunohistochemistry was used to quantify tumor angiogenesis. RESULTS: FLT3 expression is down-regulated in pancreatic cancer. Activating FLT3 mutations (ITD, D835) were not detectable in any of the pancreatic cancer cell lines. Cell growth was significantly inhibited as cell-cycle progression was reduced and programmed cell death increased. In vivo PKC412 therapy resulted in a significant inhibition of orthotopic tumor growth with abrogation of tumor angiogenesis. CONCLUSIONS: These data highlight that PKC412 may be a new compound in target therapy of inoperable pancreatic cancer patients and suggest a potential role for the combined use of broad spectrum kinase inhibitors in the management of these patients.     

Better detection of FLT3 internal tandem duplication using peripheral blood plasma DNA.

Somatic mutation of the FLT3 gene as an internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence causes constitutive tyrosine phosphorylation and activation. Tumor-specific DNA has been documented in the sera of patients with solid tumors even when it is in an early stage. We compared the detection of FLT3 ITD in DNA extracted from cells of bone marrow (BM) aspirations with DNA extracted from peripheral blood (PB) plasma in patients newly diagnosed with acute myeloid leukemia (AML; 85 patients), myelodysplastic syndrome (MDS; 16 patients), and acute lymphocytic leukemia (ALL; 16 patients). FLT3 ITD was detected in 18 (21%) AML samples and in one (6%) MDS sample in both cellular and plasma DNA but in none of the ALL samples. Hemizygous/homozygous FLT3 ITD was detected in five (28%) of the FLT3 ITD-positive AML using plasma DNA, whereas only four of these cases showed hemizygous/homozygous FLT3 ITD using cellular DNA. The presence of FLT3 ITD was associated with significantly shorter survival (P = 0.02) when only patients younger than 50 years of age (48 AML+MDS patients) were considered. This finding was independent of cytogenetics in this age group. However, patients with the FLT3 ITD hemizygous/homozygous phenotype had even shorter survival (P = <0.001). As expected, the presence of FLT3 ITD correlated with higher white blood cell (WBC) counts. These data demonstrate that plasma DNA is a reliable alternative resource for detecting FLT3ITD, especially the hemizygous/homozygous genotype. Furthermore, the data derived from this study support the notion that the presence of FLT3 ITD in conjunction with the absence of the wild-type FLT3 allele predicts an especially poor prognosis for patients with AML.     

The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia

Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC₅₀=6 nℳ), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC₅₀ 1 μℳ). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).     

Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples.

In acute myeloid leukemia (AML), activating mutations in the fms-like tyrosine kinase 3 (FLT3) gene predict poor prognosis. We determined FLT3 internal tandem duplications (FLT3/ITD) and D835 point mutations in paired initial and relapse samples from 80 pediatric and adult AML patients. One D835 point mutation was found in an initial pediatric AML sample. Fms-like tyrosine kinase 3/ITDs were present in 21 initial and 22 relapse samples (26.3 and 27.5%, respectively). Interestingly, FLT3/ITD positivity was related to a significantly shorter time to relapse, most pronounced when the ITD-positive status was found at relapse (P<0.001). However, FLT3/ITD status changed between diagnosis and relapse in 14 cases. In four patients, the FLT3/ITD became undetectable at relapse in five patients FLT3/ITDs were only detected at relapse, and in five patients the length or number of FLT3/ITDs changed. Gain of FLT3/ITDs may suggest oligoclonality with selective outgrowth of the FLT3/ITD-positive clone, whereas losses may reflect ITDs in the more mature leukemic cells rather than in the leukemic stem cell, or, alternatively, that other genetic aberrations provided a greater selective advantage. Studying FLT3/ITD kinetics in minimal residual disease setting may provide some answers for the changes we observed. Fms-like tyrosine kinase 3/ITD is a relevant marker for prognosis, and remains an important target for therapeutic inhibition.