Sigma Aldrich Israel Ltd. (Rehovot, Israel) provided most of the chemicals, including cobalt chloride (CoCl2). The text includes specific sources for various compounds. 2-Methoxyestradiol (2ME2) was purchased from Cayman Chemical (Ann Arbor, MI, USA), Everolimus (Afinitor) and kinase inhibitors (Imatinib, Ruxolitinib, and Crizotinib) were obtained from Selleck Chemicals LLC (Houston, TX, USA).

The American Type Culture Collection (ATCC, VA, USA) provided the human CML cell lines K562 and BV173. The cells were cultured in complete RPMI 1640 media supplemented with 0.1 mg/mL streptomycin, 100 units/mL penicillin, 1 % (w/v) l-glutamine, and 10 % (w/v) fetal bovine serum (Biological Industries, Israel). All cells were maintained in a humidified incubator with 5 % CO2 at 37 °C.

Using a retrovirus harboring HA-HIF1α P402A/P564A (Addgene plasmid #1900 [14]), K562 cells were stably transfected to overexpress HIF1α, as previously reported [15]. In summary, puromycin selection was used to isolate stable transfected cells after K562 cells were infected with the retrovirus. Overexpression of HA-HIF1α was detected in the transfected cells using immunoblotting with anti-HIF1α antibodies.

Using a previously established CRISPR-Cas9 method, JAK2 expression was suppressed in K562 cells [15]. In summary, lenti-cas9blast (Addgene plasmid #52,962), gRNA JAK2 (Addgene plasmid #75,728), and lentiviral packaging plasmids (pcMV-dR8.2 dvpr and pcMV-VSV-G) (Addgene plasmids #8455 and #8454) [16] were co-transfected into HEK293T cells (1.5 × 105 cells/mL) using Fugene 6 transfection reagent (Roche Applied Science, Penzberg, Germany) according with the manufacturer's instructions. The supernatant containing the lentiviral particles was collected 48 h post-transfection and utilized to infect K562 cells. Then, stably transduced clones with decreased JAK2 expression were isolated using blasticidin and puromycin selection. Western blotting was performed to confirm the downregulation of JAK2 protein levels.

The trypan blue exclusion assay was used to assess cell viability. K562 cells were seeded at a density of 2 × 105 cells per well in six-well plates. Cells were subjected to the specified treatments following a 24-hour incubation period. Dimethyl sulfoxide (DMSO) at a concentration of 1 % (w/v) was applied to the control samples. After exposure for 72 h, cells were collected, stained with a 0.4 % (w/v) trypan blue solution diluted 1:1 with culture media, and then manually counted using a hemocytometer according to the previous described protocol.

Phosphatase inhibitors (AG Scientific, CA, USA) were used to produce cell lysates. After lysing the cell pellets in the buffer for half an hour on ice, the supernatants were separated by centrifugation. The DC™ Protein Assay (Bio Rad, USA) was used to determine the protein concentration in the supernatants (absorbance measured at 630 nm). Equal protein concentrations (50–60 µg each sample) were electrophoretically separated after being loaded onto 8–12 % polyacrylamide gels. After that, the proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell BioScience GmbH, Germany). To decrease non-specific antibody binding, the membranes were then blocked using 5 % non-fat dried milk in Tris-buffered saline with Tween 20 (TBS-T).

To detect the presence of phospho-Abl (Tyr 245) and cleaved PARP (Poly(ADP-ribose) Polymerases - Cell Signaling Technology, MA, USA), α-tubulin (Santa Cruz Biotechnology, TX, USA), and phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, MA, USA), primary antibodies were employed. Every primary antibody incubation was carried out following the instructions recommended by the manufacturer.

Species-specific secondary antibodies combined with horseradish peroxidase (HRP), such as anti-mouse (NB7539, Novus Biologicals, CO, USA) and anti-rabbit (#7074, Cell Signaling Technology), were used to detect the bound primary antibodies. The SuperSignalTM West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, MA, USA) was used following the manufacturer's instructions to accomplish chemiluminescent detection [17,18].

The expressions of CD45 and CD44 on the cell surface were evaluated by flow cytometry, as previously reported [15]. In short, K562 cells were resuspended at a density of 1 × 107 cells/mL in phosphate-buffered saline (PBS) containing 0.5 % bovine serum albumin (BSA), together with K562/HIF1α and K562/Si JAK2 variations. The CD45 (KRO, #A96416, Beckman Coulter) and CD44 antibodies (FITC, #9011–0441, eBioscience) were either employed at the dilutions suggested by the manufacturer or titrated in advance to maximize staining. For forty minutes, the cell-antibody combination was incubated on ice. The labeled cells were analyzed using a Beckman Coulter Navios flow cytometer after washing with PBS mixed with 0.5 % BSA.

Using Tri Reagent (Sigma), total RNA was isolated from cells following a previously described procedure [15]. The isolated RNA was then subjected to reverse transcription (RT), which produced single-stranded cDNA. In short, 15 µL of nuclease-free water (DEPC-treated) and 1 µL of oligo(dT)17 primer were added to 1 µg of RNA, and the mixture was pre-incubated for 10 min at 70 °C before being quickly cooled on ice. Second, 2 mL of 5X AMV RT reaction buffer, 2 µL of dNTP mix (25 mM), 28 units of RNasin ribonuclease inhibitor, 30 units of AMV reverse transcriptase, and nuclease-free water were added to the annealed primer-template mixture for a final volume of 10 µL. The RT reaction was incubated for 60 min at 42 °C.

A commercial PCR kit (Bioline, Taunton, MA, USA) was used to amplify the generated cDNA. Specific primers for each gene of interest are specified in Table 1. Thirty-five amplification cycles (each cycle: 94 °C denaturation for 30 s, 55–60 °C annealing for 30 s, and 72 °C extension for 2 min and 30 s) proceeded after the first denaturation step at 94 °C for two minutes. The cycling program concluded with a last extension step that lasted 15 min at 72 °C. Primers specific to the housekeeping gene β-actin were used as a control. The PCR products (5 µL) were separated by electrophoresis in a 1.5 % agarose gel.

Quantitative RT-PCR amplification of mRNA transcripts was performed using the resultant cDNA. Each 15 µL reaction mixture included 1X SYBR GREEN reaction mix (Kappa Biosystems, Wilmington, MA, USA) and 0.2 pmol/µL of forward and reverse primers. A spectrofluorometric Rotor-Gene 6000 thermal cycler (Corbett Research, Mortlake, Australia) was used to perform the PCR. The Taq DNA polymerase was first activated by a 10-minute denaturation stage at 95 °C, which was followed by 55 cycles of denaturation at 95 °C for 15 s and a one-minute combined annealing/extension step at 55–60 °C. A range of cDNA template concentrations were evaluated to guarantee amplification within the linear range. As an internal control, the housekeeping gene β-actin was co-amplified in the same PCR reaction. The primer sequences employed in the semi-quantitative RT-PCR were the same.

A clonogenic experiment was used to evaluate the capacity of cells to form colonies on a semi-solid medium, as previously reported [19]. In summary, K562 cells were diluted in complete RPMI 1640 media with 10 % fetal bovine serum (FBS) until they reached a density of 1 × 104 cells/mL. The cell suspension was mixed with an equivalent amount of 0.6 % agar solution to reach a final agar concentration of 0.3 %. The cell-agar mixture was plated in 12-well plates over a layer of bottom agar that had already hardened and left to solidify. To maintain agar hydration after the top layer solidified, 1 mL of new medium containing the specified treatments was applied to each well. The cells were cultured for two weeks at 37 °C in a humidified environment with 5 % CO2 to enable colony formation. A colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used to visualize and quantify colonies. The plates were incubated for 4 h with 5 mg/mL MTT solution, followed by the extraction of the formazan product using a solubilization buffer (20 % SDS, 50 % N,N-dimethylformamide, and 25 mM HCl). Using a plate reader, the optical density of the extracted dye was measured with a plate reader at 570 nm with a reference wavelength of 630 nm.

A hypoxic atmosphere was created using a BioSpherix OxyCycler system (BioSpherix, Redfield, NY, USA). Cells were cultured in a custom-designed, computer-controlled incubator attached to the OxyCycler. A controlled environment of 37 °C, 5 % CO2, and 2 % O2 was maintained in the hypoxic chamber. The cells in the control group were cultured for the duration of the experiment in normoxic conditions (∼21 % O2 and 5 % CO2). Twelve hours was the incubation time for both normoxic and hypoxic experiments.

The cellular expression and location of phospho-c-Abl and HIF-1α proteins in K562 cells were determined by immunofluorescence. The immunofluorescence protocol involved coating a 96-well plate with poly-l-lysine (Sigma Aldrich Israel Ltd, Rehovot, Israel) four hours before the experiment completion and allowing it to dry for two hours. Following the seeding of 5 × 105 cells in the coated wells, they were incubated for a further two hours under normoxic conditions and then 12 h of normoxic/hypoxic conditions as described above.

Following incubation, cells were washed twice with PBS and fixed with 4 % paraformaldehyde in PBS for 20 min at room temperature (RT). Following fixation, cells were washed twice with PBS and permeabilized using a solution containing 3 % FBS and 0.1 % Triton X-100 in PBS for 1 h at room temperature. Blocking was carried out for an extra one hour at room temperature using the same permeabilization solution. The primary antibody of interest (HIF1α or phospho-c-Abl) was then diluted in 3 % FBS and 0.1 % Triton X-100 in PBS. Cells were incubated overnight after being thoroughly washed twice with 3 % FBS in PBS. DAPI (nuclear stain) and Alexa Fluor-688 (red) (Thermo Fisher Scientific. Waltham, MA, USA), a secondary antibody, were added to the cells and incubated for 30 min the next day. The last washing step was three washes using 0.1 % Triton X-100 in 3 % FBS and PBS.

Image Acquisition used a 20x magnification with images being taken with the Zeiss Cell Discoverer 7 Imaging System. The picture capture program was Zen Blue 3.7.

Protein Expression Quantification: Immunofluorescence data were used to quantify the relative amounts of phospho-c-Abl and HIF-1α protein expression.

Data were analyzed using Student's t-test. Statistical significance was set at a *p-value < 0.01 and **p-value < 0.001.

CoCl2, a known inducer of HIF1, was used to simulate hypoxic conditions in the K562 and BV173 CML cell lines [22]. Immunofluorescence analysis showed that K562 cells exposed to 100 μM CoCl2 exhibited increased levels of HIF1 comparable to exposure to low oxygen (Figure 1A and B). Real-time PCR was also used to monitor the expression of HIF1-responsive genes such as VEGF and Bcl2 (Figure 1C) and to demonstrate the functionality of induced HIF1 in K562 cells treated with CoCl2.

Figure 1.

Expression of HIF1α and pAbl in K562 under hypoxic conditions. K562 cells were treated with 100 μM cobalt chloride (CoCl2) or grown under low oxygen conditions (2 %) for 12 h. (A) Induction of HIF1α in K562 cells by treatment with CoCl2 and exposure to hypoxic conditions for 12 h. (B) Quantitation of HIF1a levels under the different conditions. (C) Real-time PCR was used to determine the relative expression of Bcl2 and VEGF in relation to β-actin in K562 cells exposed to 100 μM CoCl2. Sequences of the primers used in this study are shown in Table 1. (D) Immunofluorescence assessment of pAbl levels in K562 cells grown under normoxic, and (E) quantitation. (F) Immunofluorescence assessment of pAbl levels in K562 cells grown under hypoxic conditions after 12 h of treatment, Imatinib, Ruxolitinib, Imatinib/Ruxolitinib, and Crizotinib/Ruxolitinib and (G) quantitation. *p-value ≤0.01, **p-value ≤0.001, *** p-value ≤ 0.0001. The experiment, performed in duplicate, was repeated, with consistent results observed across both independent experiments.

Exposure to CoCl2 and growth of K562 under hypoxic conditions led to an accumulation of HIF1α (Figure 1A and B), which increased the expression of VEGF and Bcl2 by more than fourfold and twentyfold, respectively (Figure 1C). Figure 1D shows that CoCl2 treatment had no effect on Abl phosphorylation in K562. The study also showed that Imatinib and Crizotinib alone or in combination with Ruxolitinib inhibited the phosphorylation of Abl under normoxic (Figure 1D and E) and hypoxic (Figure 1F and G) conditions. Interestingly, pAbl levels were lower in hypoxic conditions (Figure 1E) compared to normoxic conditions (Figure 1G).

Next, how Imatinib and Crizotinib affected the proliferation of K562 cells in the presence and absence of CoCl2 was examined. Figure 2A shows that exposure to imatinib inhibited the proliferation of K562 in the presence or absence of CoCl2. In addition, a significant inhibition of cell proliferation in K562 cells treated with Crizotinib was observed. However, moderate drug resistance was observed in the presence of CoCl2 in Crizotinib-treated K562 cells. This suggests that the presence of CoCl2 blocks the inhibition of cell proliferation mediated by Crizotinib but not by Imatinib (Figure 2A). Signal Transducer and Activator of Transcription 3 (STAT3) has been associated with hypoxia-induced chemoresistance of ovarian cancer cells [23]. Therefore, the JAK1/2 inhibitor Ruxolitinib [24], which regulates the activity of STAT3, was used to investigate the role of JAK1/2 in the sensitivity of CML cells to Crizotinib. We wondered whether the addition of Ruxolitinib to Crizotinib in the presence of CoCl2 could restore sensitivity. Treatment with Ruxolitinib alone had no effect on the proliferation of K562 cells in the presence or absence of CoCl2 (Figure 2B). Furthermore, there was no significant difference in the inhibition of K562 cell proliferation between the combinations of Imatinib and Ruxolitinib (Figure 2B). Interestingly, the combination of Ruxolitinib and Crizotinib did not restore the sensitivity of K562 to Crizotinib. In contrast, the inclusion of Ruxolitinib increased Crizotinib resistance in K562 cells (Figure 2B). Comparable data were also obtained using other CML cells such as BV173 (Figure 2C).

Figure 2.

Effect of cobalt chloride (CoCl2) and ruxolitinib on the sensitivity of K562 and BV 173 cells to treatment with Imatinib or Crizotinib. K562 or BV 173 cells exposed to 100 µM CoCl2 and treated with 1 % DMSO, 1 μM Imatinib, Crizotinib or Ruxolitinib for 12 h. (A) Measurement of the viability of K562 with the trypan blue exclusion assay using a two-sample t-test. Immunoblot of (B) K562 and (C) BV 173 cells exposed to 1 µM Imatinib, Crizotinib, and Ruxolitinib in the presence or absence of 100 µM CoCl2. Filters were probed with anti-c-PARP and α-tubulin antibodies. The numbers below the plot represent the relative expression levels normalized to α-tubulin.

The above data suggests that the sensitivity of CML cells to Crizotinib is dependent on JAK1/2 activity and hypoxic conditions (Figure 2). However, neither hypoxia nor JAK1/2 activity had a significant effect on sensitivity to Imatinib (Figure 2). HIF1α is an important mediator of hypoxic conditions and modulates the transcription of many genes. To directly demonstrate the role of hypoxia and JAK2 in regulating sensitivity to Crizotinib, a stable mutant of HIF1α (Pro 402 and Pro 564 were replaced by Ala) [25] was overexpressed in K562. Under normoxic conditions, the HIF1a mutant Pro 402 Ala, Pro 564 Ala is not hydroxylated and is therefore not recognized by the von Hippel-Lindau tumor suppressor protein (VHL) for proteasome degradation [26]. Figure 3A shows that HA-HIF1α is overexpressed in K562/HIF1 cells under normoxic conditions. CD44 is one of the genes regulated by HIF1α [27], and monitoring of levels in K562 and K562/HIF1α cells showed upregulation of CD44 (Figure 3B).

Figure 3.

Effect of overexpression of HIF1a on the chemoresistance of K562. (A) Immunoblot of HIF1α in K562 and K562/HIF1α cells. (B) The level of expression of CD44 in K562 and K562/HIF1α was determined by FACS analysis. (C) Levels of pAbl and cleaved PARP in K562 and K562/HIF1α exposed to Imatinib (1μ M), Crizotinib (1μ M), or Ruxolitinib (1 μM) for 24 h. (D) Clonogenicity of K562 and K562/HIF1α cells under semi-solid conditions in the presence of Imatinib (0.3, 1 and 3 µM) and Crizotinib (0.3, 1 and 3 µM) in combination with Ruxolitinib (1 µM). (E) Heatmap of absorptance of dye levels extracted from stained colonies in the different samples. The experiment was performed twice, yielding comparable results.

Next, the activity of the Abl inhibitor in K562 cells stably expressing HIF1α was examined. The levels of phosphorylated Bcr/Abl and Abl were essentially unaffected by the overexpression of HIF1α, as shown in Figure 3C. However, the levels of phosphorylated Bcr/Abl and Abl in K562 and K562/HIF1α lysates were completely inhibited by Imatinib or Crizotinib alone or in combination with Ruxolitinib (Figure 3C). This suggests that the ability of Imatinib and Crizotinib to inhibit auto-phosphorylation of the Abl protein is not affected in cells overexpressing HIF1α or exposed to Ruxolitinib. When K562 cells were exposed to Imatinib, PARP was significantly cleaved. Interestingly, the combination of Ruxolitinib and Imatinib decreased the amount of cleaved PARP in K562/HIF1α cells but not in K562 cells. The amount of cleaved PARP was almost twice as high in Crizotinib-treated K562 cells compared to Imatinib-treated cells. In addition, the combination of Ruxolitinib and Crizotinib significantly reduced the amount of cleaved PARP in K562 by about 50 %. In contrast, neither Crizotinib nor Crizotinib in combination with Ruxolitinib resulted in cleavage of PARP in K562/HIF1α cells. Figure 3 shows that HIF1α is responsible for the reduction of cleaved PARP levels in K562 cells when exposed to Crizotinib, although Crizotinib still inhibits Bcr/Abl activity. In addition, it supports the notion that JAK1/2 inhibition contributes to the reduced sensitivity of Imatinib and Crizotinib.

A comparison of the effect of Imatinib and Crizotinib was also evaluated using a clonogenicity assay. Figure 3D shows that Imatinib inhibited colony formation in K562 and K562/HIF1α cells at concentrations of 1 and 3 μM. Crizotinib was more effective in inhibiting colony formation of K562, and significant inhibition was observed at the lowest concentration (0.3 μM). The ability of Crizotinib to inhibit K562/HIF1α was impaired, indicating some degree of resistance. Interestingly, the addition of Ruxolitinib reduced the ability of Crizotinib to inhibit colony formation in both K562/HIF1-positive and -negative cells. Of note, the combination of Ruxolitinib with Crizotinib showed a minimal effect on the proliferation of K562 (Figure 2A - cells grown in two-dimensional [2D] culture) and a significant effect on K562 treated with Crizotinib plus Ruxolitinib in a three-dimensional (3D) culture (Figure 3D).

The JAK2 gene was also silenced in K562 cells (Figure 4A). The sensitivity of K562, K562/Si JAK2 and K562/ HA-HIF1α P402A/P564A to a range of Abl kinase inhibitors (AKIs) was monitored. The results shown in Figure 4B demonstrate that the different cells have different sensitivities to the different AKIs.

Figure 4.

Effect of JAK2 silencing and HIF1α overexpression on the sensitivity of K562 to different Abl kinase inhibitor treatments. (A) Immunoblot of JAK2 in K562 and K562 SiJAK2 cells. (B) Proliferation blot showing remaining K562, K562/Si JAK2, and K562 HIF1α cells when exposed to 1000 nM Imatinib, Crizotinib, ponatinib, or GNF-5. (C) Proliferation blot of the different K562 cells that were exposed to different concentrations of Crizotinib. Cell viability was monitored as described in Materials and Methods. *p-value ≤0.01, **p-value ≤0.001, *** p-value ≤ 0.0001. The experiment was run in duplicate and repeated (for a total of two independent experimental runs), with both yielding comparable outcomes.

Exposure of various K562 cells to AKIs resulted in a decrease in cell viability. When K562/HIF1α cells were treated with AKIs, a marked and reproducible increase in cell viability was observed compared to K562 cells (Figure 4B), indicating partial drug resistance due to overexpression of HIF1α (Figure 4B). The ability of Crizotinib to inhibit the proliferation of K562 cells was significantly different from that of K562/Si JAK2 and K562/HIF1α cells. Crizotinib was less effective in inhibiting the proliferation of K562/Si JAK2 and K562/HIF1α cells (Figure 4B). Thus, the percentages of viable cells in K562, K562/Si JAK2, and K562/ HA-HIF1α when exposed to 500 nM Crizotinib for 72 h were 23.4, 57.9, and 63.4, respectively (Figure 4C). In addition, no significant differences in cell viability were observed between K562 cells exposed to other AKIs, including ponatinib and GNF-5 (Figure 4B). These results suggest that the reduced sensitivity to Crizotinib is significant in cells overexpressing HIF1α or silenced with JAK2, whereas it is less evident with other AKIs.

The data of this study suggest that HIF1α is responsible for mediating Crizotinib chemoresistance in CML cells. Therefore, it investigated whether HIF1α modulators can restore sensitivity to Crizotinib. Two HIF1α modulators, 2ME2 and Everolimus (Afinitor), were used. 2ME2, a natural estradiol metabolite, has been shown to inhibit the nuclear accumulation and activity of HIF1α in an oxygen- and proteasome-independent manner [28], while Afinitor, an mTOR inhibitor, has been shown to reduce the expression of HIF1α and restore chemosensitivity in ALL cells [29].

Figure 5A shows that Crizotinib induces PARP cleavage in K562 cells but not in K562/HIF1α cells. However, 2ME2 was only marginally active in inducing PARP cleavage in K562 cells, whereas Afinitor was not. The combination of Afinitor with Crizotinib had little effect on the sensitivity of K562 cells to Crizotinib, particularly in K562/HIF1α cells. In contrast, the combination of 2ME2 and Crizotinib significantly increased the amount of cleaved PARP in K562/HIF1α cells, suggesting that 2ME2 can restore the sensitivity of Crizotinib in K562 cells overexpressing activated HIF1α. Thus, the decreased sensitivity of CML cells to Crizotinib under hypoxic conditions is primarily mediated by the stabilization and increased activity of HIF1α, and inhibiting the function of HIF1α may restore the sensitivity of Crizotinib in hypoxic CML cells.

Figure 5.

Effect of 2ME2 on HIF1α-mediated resistance to Crizotinib. K562 or K562/HIF1α cells were treated with 1 % DMSO, Imatinib, Crizotinib, Afinitor, or 2ME2 for 24 h. (A) Immunoblot of K562 or K562/HIF1α cells treated with 1 µM Crizotinib, Afinitor, or 2ME2. Filters were probed with anti-c-PARP and α-tubulin antibodies. The numbers below the different lanes represent relative values normalized to α-tubulin. (B and C) Relative expressions of (B) Bcl2 and (C) Mcl1 genes compared to the β-actin gene were determined by real-time PCR using K562 and K562/HIF1α cells treated with 1 % DMSO, Imatinib, or Crizotinib for 24 h. Sequences of the primers used in this study are shown in Table 1. The levels of PCR amplicons were analyzed using a two-sample t-test. The mean relative expression (log2) ± standard error of the mean is shown. *p-value ≤0.01, **p-value ≤ 0.001 and *** p-value ≤ 0.0001. The experiment was performed twice, yielding consistent results.

To explore the possible mechanism by which activation of HIF1α confers chemoresistance to CML cells despite complete inhibition of Bcr/Abl, changes in apoptosis-related gene expression were monitored in K562 and K562/HIF1α cells treated with Crizotinib. The expression of Mcl1 and Bcl2 genes in K562 and k562/HIF1α cells treated with Imatinib or Crizotinib were monitored (Figure 5B). Treatment of K562 cells with Imatinib resulted in a small reduction in the levels of both genes. Treatment with Crizotinib was more effective in reducing the levels of the MCL1 and Bcl2 genes. In contrast, treatment with Imatinib in K562/HIF1α led to a similar reduction in the levels of both genes as in K562 cells. However, the levels of Bcl2 and MCL1 were significantly higher in Crizotinib-treated K562/HIF1α cells than in Imatinib-treated cells. This suggests that the levels of the two anti-apoptotic genes Bcl2 and MCL1 are relatively higher in K562/HIF1α exposed to Crizotinib compared to Imatinib treatment and likely contribute to the chemoresistance to Crizotinib observed in CML cells under hypoxic conditions.