Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis

Abstract

Hematopoietic cells normally require cell extrinsic signals to maintain metabolism and survival. In contrast, cancer cells can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells receive insufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and the self-digestive process of autophagy is elevated to degrade bulk cytoplasm and organelles. Although autophagy has been proposed to provide a cell-intrinsic nutrient supply for mitochondrial oxidative metabolism and to maintain cellular homeostasis through degradation of damaged organelles or protein aggregates, its acute role in growth factor deprivation or inhibition of oncogenic kinases remains poorly understood. We therefore developed a growth factor-dependent hematopoietic cell culture model in which autophagy can be acutely disrupted through conditional Cre-mediated excision of the autophagy-essential gene Atg3. Treated cells rapidly lost their ability to perform autophagy and underwent cell cycle arrest and apoptosis. Although Atg3 was essential for optimal upregulation of mitochondrial oxidative pathways in growth factor withdrawal, this metabolic contribution of autophagy did not appear critical for cell survival, as provision of exogenous pyruvate or lipids could not completely rescue Atg3 deficiency. Instead, autophagy suppressed a stress response that otherwise led to p53 phosphorylation and upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma. Importantly, BCR-Abl-expressing cells had low basal levels of autophagy, but were highly dependent on this process, and rapidly underwent apoptosis upon disruption of autophagy through Atg3 deletion or treatment with chemical autophagy inhibitors. This dependence on autophagy extended in vivo, as Atg3 deletion also prevented BCR-Abl-mediated leukemogenesis in a cell transfer model. Together these data demonstrate a critical role for autophagy to mitigate cell stress, and that cells expressing the oncogenic kinase BCR-Abl appear particularly dependent on autophagy for cell survival and leukemogenesis.

Figure 1. Autophagy can be acutely disrupted by inducible excision of Atg3 in the hematopoietic A3C cell population

A. Generation and model of immortalization, culture conditions, and Atg3 excision from Atg3^F/F Rosa26-Cre-ER mouse of A3C cells. B. A3C cells were cultured with ethanol or 0.5 μM 4-hydroxytamoxifen (4OHT) for the indicated times, and loss of Atg3 was observed by immunoblot. A non-specific band is indicated by <. C.,D. Bcl-2-expressing A3C cells were cultured with ethanol or 4OHT for 4 days, then cultured in the absence (C) or presence (D) of 40 μM chloroquine (CQ) for 10 hours, and LC3-II, GABARAP, Gate-16, and p62 protein accumulation were assessed by immunoblot. LC3 antibody used preferentially recognized LC3-II. E. A3C GFP-LC3-expressing cells were cultured with ethanol or 4OHT for two days and subjected to analysis on the Amnis ImagestreamX Multispectral Imaging Flow Cytometer for LC3-puncta. Left panel shows histogram of the number of LC3-puncta in individual cells, right panel shows examples of more punctate vs less punctate patterns. Data shown are representative of three or more experiments.

Figure 2. Disruption of autophagy causes cell cycle arrest in culture

A. NGF-expressing A3C control cells were cultured in the presence or absence of 4OHT, and cell number was assessed daily using a Coulter Z2 Particle Counter. B,C. Bcl-2-expressing A3C cells were cultured with ethanol or 4OHT for three days, with media being changed and new drug being added on day two, then cultured with Bromo-2-deoxyuridine (BrdU) and BrdU incorporation and PI staining (to indicate DNA content) were assessed by flow cytometry for (B) BrdU incorporation and (C) PI cell-cycle analysis. Means and standard deviations of triplicate samples are shown. Asterisks denote p < 0.05 by Student’s t-Test of 4OHT-treated samples compared to control-treated samples.

Figure 3. Autophagy disruption delays atrophy and enhances cell death in both the presence and absence of growth factor

A. Bcl-2-expressing cells were cultured for four days in the presence or absence of 4OHT to delete Atg3, then cultured in the presence or absence of both 4OHT and IL3. Cell size of Bcl-2-expressing cells was assessed by forward scan flow cytometry. B. Control NGF or Bcl-2-expressing A3C cells were cultured for two days with ethanol or 4OHT, then cultured in the presence or absence of both 4OHT and IL3, and survival of control NGF and Bcl-2-expressing cells was assessed by propidium iodide exclusion flow cytometry. Note different time scales. C. Bcl-2-expressing A3C cells were cultured for two days with ethanol or 4OHT to prevent autophagy, and withdrawn from IL3 for indicated times. Mcl-1, Puma, Bim, and Bax, were analyzed by immunoblot. Means and standard deviations of triplicate samples are shown. Data shown are representative of three or more experiments. Asterisks denote p < 0.05 by Student’s t-Test of 4OHT-treated samples compared to control-treated samples.

Figure 4. Atg3 deletion affects cell metabolism, but these changes do not appear critical for survival early in growth factor withdrawal

A-C. Bcl-2-expressing A3C cells were cultured with IL3 or withdrawn from IL3 for 24 hours and (A) glycolysis, (B) intracellular lactate, and (C) short-chain C2 (acetyl) and the long-chain acyl carnitines C16 (palmitate), C18:1 (oleate) and C18 (stearate) were measured. D-F. Control NGF (D), Bcl-2-expressing (E), or Glut1-expressing (F) were cultured for two days with either ethanol or 4OHT to inhibit autophagy, then cultured in the presence or absence of IL3 along with a 1:1 mixture of 5 mM oleate and palmitate (OP), 10 mM methyl-pyruvate (MeP), OP + MeP, or BSA as a control, and cell viability was assessed. Note different time scales. Means and standard deviations of triplicate samples are shown, standard errors for mass spectrometry experiments. Data shown are representative of three or more experiments. Asterisks denote p < 0.05 by Student’s t-Test of IL3-withdrawn samples compared to control samples for B,C, 4OHT-treated samples compared to control-treated samples for F.

Figure 5. Atg3 deletion leads to cell stress and p53 activation

A,B. Bcl-2-expressing A3C cells were cultured in the presence or absence of 4OHT for two days followed by withdrawal from IL3 for indicated times. (A) The ER stress markers Chop, BiP, and Calnexin were analyzed by immunoblot. Some cells were treated with 2 μg/mL tunicamycin (Tun) for 12 hours. (B) Phospho-p53 serine 18, total p53, p21, and the DNA damage response protein γH2A.X (phospho histone H2A.X serine 139) were analyzed by immunoblot. C. FL5.12 cells were withdrawn from IL3 for the indicated times, and γH2A.X and p21 were analyzed by immunoblot. Data shown are representative of three or more experiments.

Figure 6. BCR-Abl-transformed A3C cells are sensitive to loss of autophagy

A. BCR-Abl-expressing A3C cells were cultured for two days in the presence or absence of 4OHT and 0.1 μM imatinib, and survival was assessed. B,C. Control NGF and BCR-Abl-expressing cells were cultured in the presence or absence of both IL3 and 4OHT for ten days, with media being changed and new 4OHT added on day two, and growing cells being split every two days. (B) Cell accumulation over time was measured by quantitation on a Coulter Z2 Particle Counter, and (C) death was measured by flow cytometry. D. Control NGF and BCR-Abl-expressing cells were treated with 4OHT for the times indicated, and phospho-p53 serine 18, total p53, p21, Puma, and γH2A.X were analyzed by immunoblot. Means and standard deviations of triplicate samples are shown. Data shown are representative of three or more experiments. Asterisks denote p < 0.05 by Student’s t-Test of 4OHT-treated samples compared to control-treated samples.

Figure 7. Pharmacologic inhibition of autophagy promotes death of BCR-Abl-transformed A3C cells

A,B. Control NGF and BCR-Abl-expressing cells were cultured in the presence or absence of both IL3 and 10 μM chloroquine (CQ), with media being changed, new 4OHT being added, and growing cells being split on day two. (A) Cell accumulation over time was measured by quantitation on a Coulter Z2 Particle Counter, and (B) death was measured by flow cytometry. C,D. Control NGF and BCR-Abl-expressing cells were cultured in the presence of IL3 or absence of IL3 for BCR-Abl-expressing cells, and with 0, 5, or 10 mM 3-methyladenine (3MA). (A) Cell accumulation over time was measured by quantitation on a Coulter Z2 Particle Counter, and (B) death was measured by flow cytometry Means and standard deviations of triplicate samples are shown. Data shown are representative of three or more experiments. Asterisks denote p < 0.05 by Student’s t-Test of 4OHT-treated samples compared to control-treated samples.

Figure 8. shRNAi targeting of p53 alleviates some effects of Atg3 excision

A. BCR-Abl-expressing cells stably transduced with a control vector or p53 shRNAi were treated with 4OHT for the indicated times, and phospho-p53 serine 18, total p53, p21, Puma, and γH2A.X were analyzed by immunoblot. Quantitations are provided for p21, Puma, and γH2A.X, normalized to relative background and to actin. B,C. BCR-Abl-expressing cells with or without p53 shRNAi were treated with 4OHT for the indicated times, with media being changed, new 4OHT added, and growing cells being split on day two. (B) Cell accumulation over time was measured by quantitation on a Coulter Z2 Particle Counter, and (C) death was measured by flow cytometry. Data shown are representative of three or more experiments. Asterisks denote p < 0.05 by Student’s t-Test of 4OHT-treated samples compared to control-treated samples.

Figure 9. Loss of autophagy prevents formation of BCR-Abl⁺ cancer in an allograft model of leukemia

A. Immunocompromised mice were injected with control or BCR-Abl-expressing A3C cells and mice received intraperitoneal injections vehicle or tamoxifen for three days and time of disease-free survival was measured. B-C. Spleens were removed from each animal and (B) weighed, with representative spleens are shown in the bottom panel and (C) subject to histological examination with hematoxylin and eosin. Means and standard deviations are from six mice each for injections with BCR-Abl-expressing cells, and four mice each for injections with NGF-expressing cells. Asterisks denote p < 0.05 by Student’s t-Test. D. Immunocompromised mice were injected with GRP+ or BCR-Abl A3C cells and mice received intraperitoneal injections vehicle or tamoxifen for three days either immediately after cell injection, or three consecutive days two weeks after cell injection, as indicated. Mice were sacrificed at 50 days after cell injection, and individual spleens were weighed.