Autophagy activation mediates resistance to FLT3 inhibitors in acute myeloid leukemia with FLT3-ITD mutation

Sorafenib (CAS #475207-59-1) and quizartinib (AC220, CAS #950769-58-1) were purchased from Selleck. Chloroquince (CQ, CAS #50-63-5) was bought from Sigma. The antibodies against human-phosphorylated (p)-p44/42 MAPK (ERK1/2, Thr202/Tyr204, CAS #4370), p-FLT3 (Tyr589/591, CAS ##60413), p-mTOR (Ser2448, CAS #5536), p-S6K (Ser240/244, CAS #2215), mTOR (CAS ##2983), S6K (CAS #9202), Beclin-1 (CAS #4122), LC3B (CAS #3868), ATG5 (D5G3, CAS #9980), p62/SQSTM1 (CAS #5114), c-Myc (D84C12, CAS #5605) and cleaved caspase-3 (Asp175, CAS #9661) were purchased from Cell Signaling Technology. Against ERK2 (CAS #sc-1647) and FLT3 (CAS #sc-19635) were from Santa Cruz Biotechnology. Anti-GAPHD (CAS #G9545) was purchased from Millipore Sigma.

Six patients with FLT3-ITD-positive AML were included from the ClinicalTrials (NCT02474290). The detail of clinical characteristics and treatment protocol had been reported [14]. Bone marrow samples were obtained from those patients at diagnosis, continued complete response (CCR) or relapsed after written informed consents were gotten according to the institutional guidelines of Medicine Institutional Review Boards of Nanfang Hospital, Southern Medical University. The mononuclear (MNC) cells in these samples were purified by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation, and then cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS).

The Ba/F3-ITD, Ba/F3-D835Y and Ba/F3-ITD + D835Y cell lines, and the human AML cell line MOLM14 were all kindly provided by professor Andreeff Michael (Department of Leukemia Research, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX) in 2016. All cell lines were validated by short tandem repeat (STR) DNA fingerprinting using the AmpFISTR Identifiler Kit as described before [15]. All cells were maintained in RPMI medium supplemented with 10% FCS.

Cell viability was assessed using the trypan blue dye exclusion method, and apoptosis was determined via flow cytometry (FACS) by Annexin V positivity as described [16].

For measuring apoptosis induction in the leukemia cells co-cultured with MSCs, the cells were trypsinized and stained with CD90-PE, CD45-APC and Annexin V-FITC (all from BD Biosciences), and apoptosis was assessed by measuring Annexin V-FITC positivity after excluding the CD90 + CD45—(used as a MSC marker) cell population.

According to our previous report [17], samples were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, then washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate buffered tannic acid, postfixed with 1% buffered osmium, and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 60 ℃ oven for approximately 3 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 80 kV. Micrographs were taken at 7500 × or 50,000 × magnification.

The cells were treated with the indicated agents and then collected in lysis buffer. Phosphorylation and total protein levels were determined using Odyssey Infrared Imaging System (LI-COR Biosciences).

The data are presented as the means ± SD of triplicate samples or assays. The statistical analyses were performed using unpaired Student t test. A 2-sided Fisher exact test was used to determine statistical significance between different groups. A P value ≤ 0.05 was considered statistically significant.

Six patients with newly diagnosed FLT3-ITD-positiver AML, of whom 2 were CCR and 4 relapsed on sorafenib treatment, were included and isolated MNC cells from bone marrow at diagnosis and the status of CCR or relapse. The characteristics of the 6 patients were presented in Table 1. The primary AML cells, being isolated from 4 relapsed patients (Case #3 and #5 with FLT3-ITD mutation, case #4 with FLT3-ITD + D835Y mutation, and case #6 without FLT3 mutation at relapse) were truly resistant to sorafenib in vitro (Fig. 1A–D). In order to assess the expression of autophagy in sorafenib-resistant cells, we detected autophagy makers in those primary cells after vs. before sorafenib treatment. Immunoblotting analyses showed that, in comparison with the results before the treatment, LC3B-II/I ratio and ATG5 expression increased, and p62 degraded in the sorafenib-resistant blasts. In contrast, LC3B-II/I ratio and ATG5 expression decreased, and p62 accumulated in the sorafenib-sensitive cells (Fig. 1C), suggesting that FLT3 inhibitor resistant primary FLT3-ITD-positive AML cells might overexpress autophagy.

In order to test activation of autophagy in sorafenib-resistant AML cells, sorafenib-resistant cell lines were built such as Baf3 cells with FLT3-D835Y or FLT3-ITD + D835Y mutation and detected the expression of autophagy markers including LC3B, Beclin-1, ATG5 and p62. In line with previous report [18], Ba/F3-ITD + D835Y mutant cells were resistant to sorafenib (Fig. 2A). Compared with Ba/F3-ITD mutant cells, which was sensitive to sorafenib (Fig. 2A), both Ba/F3-D835Y and Ba/F3-ITD + D835Y mutant cells presented higher expression of LC3B-II, Beclin-1 and ATG5, and degratation of p62 (Fig. 2B), indicating that sorafenib-resistant FLT3-ITD-positive cell lines could also overexpress autophagy and the cytoprotective autophagy might be activated by acquired resistant mutation..

The data above showed that sorafenib-resistant leukemia cells expressed higher autophagy, suggesting autophagy activation might be associated with FLT3 inhibitor resistance in FLT3-ITD-positive AML. To test this hypothesis, we used CQ to down-regulate autophagy in sorafenib-resistant cells, then detected whether it would strengthen the anti-leukemia effect of sorafenib. As mention above, Ba/F3-ITD + D835Y cells were resistant to sorafenib. After inhibition of autophagy with CQ, Ba/F3-ITD + D835Y cells were turned to be sensitive to sorafenib treatment (Fig. 3A). In line with this, western blot showed sorafenib increased the expression of cleaved-caspase 3 in Ba/F3-ITD + D835Y cells after being dealt with vs. without CQ (Fig. 3B). In addition, inhibition of autophagy with CQ also enhanced the anti-leukemia effect of sorafenib in Ba/F3-ITD cells (Fig. 3A and B). Furthermore, sorafenib-resistant primary AML cells with FLT3-ITD mutation (case #3, Fig. 3C and D) or FLT3-ITD + D835Y mutation (case #4, Fig. 3E and F) were also sensitized to sorafenib after being dealt with CQ. Taking together, sorafenib-resistant leukemia cells overpresented autophagy markers; inhibition of autophagy partly overcame sorafenib resistance, suggesting activation of autophagy could be an important factor for FLT3 inhibitor resistance in FLT3-ITD-poisitive AML cells.

We then went further to explore how sorafenib worked after autophagy was inhibited. Immunoblotting data showed that, in Ba/F3-ITD + D835Y cells, without CQ co-treatment, sorafenib could only obviously down-regulate the expression of p-FLT3, but not FLT3 downstream signaling, and no apparent pro-apoptotic affect was induced. However, after being dealt with CQ, autophagy was inhibited, sorafenib significantly suppressed the downstream signaling of FLT3 and markedly induced the expression of cleaved caspase-3 (Fig. 4B), which accorded with autophagy inhibition enhancing the killing effect of sorafenib in Ba/F3-ITD + D835Y cells. In accordance with that, in sorafenib-sensitive cell line Ba/F3-ITD, after autophagy was inhibited with CQ, FLT3 downstream signaling was more markedly suppressed and cleaved caspase-3 was significantly induced by sorafenib (Fig. 4A). All these data indicated that autophagy might bypass activate FLT3 downstream signaling to decrease the cytotoxic effect of FLT3 inhibitor in FLT3 inhibitor resistant even sensitive leukemic cells.

It is reported that BME could up-regulate autophagy to mediate chemotherapy resistance in AML [19]. Whether BME-mediated autophagy could induce FLT3 inhibitor resistance in FLT3-ITD-positive AML remains unsure. To test the affect of BME on activation of autophagy and mediation of FLT3 inhibitor resistance in FLT3-ITD mutant cells, we detected the expression of autophagy markers and the anti-leukemia effect of FLT3 inhibitors including sorafenib and AC220 in FLT3-ITD-mutated cells with vs. without MSCs (case #3) co-culture.

After co-culture with MSCs, no matter from the relapsed patient (case #3) or from the CCR case (case #2) with FLT3-ITD mutation, the number of phagosomes in MOLM14 cells being detected with transmission electron microscopy showed significant increase (Fig. 5A and B). In accordance with this, the expression of LC3B-II, Beclin-1 and ATG5 up-regulated, and p62 degraded with vs. without MSCs co-culture, with western blot assessment (Fig. 5C and D). In addition, MSCs from case #3 significantly decreased the killing effect of sorafenib either at the concentration of 40 nM or 80 nM in MOLM14 cells (Fig. 6A). MSCs-mediated protection on MOLM14 cells was also found in the treatment of AC220 (Fig. 7B2). In agreement with this, the immunoblotting data showed that in contrast to the result without MSCs co-culture, MSCs from case #3 upregulated the expression of LC3B-II and Beclin-1 in MOLM14 cells and weakened the inhibition efficacy of sorafenib on FLT3 signaling pathway, especially on FLT3 downstream signaling, presenting as reducing suppression on p-FLT3, p-ERK1/2 and p-mTOR with MSCs co-culture, and then decreased the expression of cleaved-caspase 3 (Fig. 6B), which could explain MSCs decreased the anti-leukemia effect of sorafenib or AC220 in MOLM14 cells, suggesting that MSCs-mediated FLT3 inhibitor resistance in FLT3-ITD mutant cells might be associated with activating autophagy.

Then we used CQ to inhibit autophagy in MOLM14 cells. After inhibition of autophagy, MSCs-mediated resistance to sorafenib (Fig. 7A2) or AC220 (Fig. 7B2) was overcome (Fig. 7A3 and B3), and even the anti-leukemia effect of sorafenib (Fig. 7A1) or AC220 (Fig. 7B1) was sensitized regardless of MSCs co-culture, in agreement with the report of FLT3-ITD up-regulating autophagy to mediate resistance to FLT3 inhibitors in AML [13]. Taking together, BME-mediated FLT3 inhibitor resistance might be associated with MSCs-inducing autophagy; Inhibition of autophagy could partly overcome BME-mediated resistance to FLT3 inhibitors, in FLT3-ITD-positive AML.