Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.     

Suberoylanilide hydroxamic acid (SAHA) causes tumor growth slowdown and triggers autophagy in glioblastoma stem cells

Although suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been used in clinical trials for cancer therapies, its pharmacological effects occur through a poorly understood mechanism. Here, we report that SAHA specifically triggers autophagy and reduces cell viability via promotion of apoptosis in the late phase of glioblastoma stem cells (GSCs). Using a cell line cultured from a glioblastoma biopsy, we investigated the properties and effects of GSCs under SAHA treatment in vitro. In vivo xenograft assays revealed that SAHA effectively caused tumor growth slowdown and the induction of autophagy. SAHA was sufficient to increase formation of intracellular acidic vesicle organelles, recruitment of LC3-II to the autophagosomes, potentiation of BECN1 protein levels and reduced SQSTM1 levels. We determined that SAHA triggered autophagy through the downregulation of AKT-MTOR signaling, a major suppressive cascade of autophagy. Interestingly, upon depletion or pharmacological inhibition of autophagy, SAHA facilitates apoptosis and results in cell death at the early phase, suggesting that SAHA-induced autophagy functions probably act as a prosurvival mechanism. Furthermore, our results also indicated that the inhibition of SAHA-induced autophagy using chloroquine has synergistic effects that further increase apoptosis. Moreover, we found that a reduced dose of SAHA functioned as a potent modulator of differentiation and senescence. Taken together, our results provide a new perspective on the treatment of GSCs, indicating that SAHA is a promising agent for targeting GSCs through the induction of autophagy.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

Sequence-specific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid

Acetylation of histones leads to conformational changes of DNA. We have previously shown that the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induced cell cycle arrest, differentiation, and apoptosis. In addition to their antitumor effects as single agents, HDAC inhibitors may cause conformational changes in the chromatin, rendering the DNA more vulnerable to DNA damaging agents. We examined the effects of SAHA on cell death induced by topo II inhibitors in breast cancer cell lines. Topo II inhibitors stabilize the topo II-DNA complex, resulting in DNA damage. Treatment of cells with SAHA promoted chromatin decondensation associated with increased nuclear concentration and DNA binding of the topo II inhibitor and subsequent potentiation of DNA damage. While SAHA-induced histone hyperacetylation occurred as early as 4 h, chromatin decondensation was most profound at 48 h. SAHA-induced potentiation of topo II inhibitors was sequence-specific. Pre-exposure of cells to SAHA for 48 h was synergistic, whereas shorter pre-exposure periods abrogated synergy and exposure of cells to SAHA after the topo II inhibitor resulted in antagonistic effects. Synergy was not observed in cells with depleted topo II levels. These effects were not limited to specific types of topo II inhibitors. We propose that SAHA significantly potentiates the DNA damage induced by topo II inhibitors; however, synergy is dependent on the sequence of drug administration and the expression of the target. These findings may impact the clinical development of combining HDAC inhibitors with DNA damaging agents.     

Proteomic analysis revealed association of aberrant ROS signaling with suberoylanilide hydroxamic acid-induced autophagy in Jurkat T-leukemia cells

Suberoylanilide hydroxamic acid (SAHA) is a newly emerging histone deacetylase inhibitor (HDACi) and has been approved in phase II clinical trials for treating patients with cutaneous T-cell lymphoma. Autophagy is a conserved self-digestion process that degrades cytoplasmic materials and recycles long-lived proteins and organelles within cells. In this study, we demonstrate that SAHA stimulates autophagy in Jurkat T-leukemia cells, which was evidenced by the appearance of autophagic vacuoles, formation of acidic vesicular organelles, recruitment of LC3-II to the autophagosomes and conversion of LC3-I to LC3-II. Moreover, SAHA treatment upregulated expression of Beclin 1 and Atg7 and promoted formation of the Atg12-Atg5 conjugate. Furthermore, inhibition of autophagy by chloroquine (CQ) enhanced SAHA-induced apoptosis. To determine the underlying mechanism of SAHA-induced autophagy, two complementary proteomic approaches (2-DE and SILAC), coupled with ESI-Q-TOF MS/MS analysis are utilized to profile differentially expressed proteins between control and SAHA-treated Jurkat T-leukemia cells. In total, 72 proteins were identified with significant alterations. Cluster analysis of the changed proteins reveal several groups of enzymes associated with energy metabolism, anti-oxidative stress and cellular redox control, which suggested an abnormal reactive oxygen species (ROS) production in SAHA-treated Jurkat T-leukemia cells. These observations were further confirmed by ROS chemiluminescence assay. Mechanistic studies revealed that SAHA-triggered autophagy was mediated by ROS production, which could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we illustrated that Akt-mTOR signaling, a major suppressive cascade of autophagy, was inactivated by SAHA treatment. Taken together, our study identifies autophagy as a reaction to counter increased ROS and is thus involved as a cellular prosurvival mechanism in response to SAHA treatment.     

Role of caspases,Bid, and p53 in the apoptotic response triggered by histone deacetylase inhibitors trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA)

Histone deacetylase activity is potently inhibited by hydroaximc acid derivatives such as suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). These inhibitors specifically induce differentiation/apoptosis of transformed cells in vitro and suppress tumor growth in vivo. Because of its low toxicity, SAHA is currently evaluated in clinical trials for the treatment of cancer. SAHA and TSA induce apoptosis, which is characterized by mitochondrial stress, but so far, the critical elements of this apoptotic program remain poorly defined. To characterize in more detail this apoptotic program, we used human cell lines containing alterations in important elements of apoptotic response such as: p53, Bcl-2, caspase-9, and caspase-3. We demonstrate that caspase-9 is critical for apoptosis induced by SAHA and TSA and that efficient proteolytic activation of caspase-2, caspase-8, and caspase-7 strictly depends on caspase-9. Bcl-2 efficiently antagonizes cytochrome c release and apoptosis in response to both histone deacetylase inhibitors. We provide evidences that translocation into the mitochondria of the Bcl-2 family member Bid depends on caspase-9 and that this translocation is a late event during TSA-induced apoptosis. We also demonstrate that the susceptibility to TSA- and SAHA-induced cell death is regulated by p53.     

Histone deacetylase inhibitors induce mitochondrial elongation

Although various stimuli-inducing cell demise are known to alter mitochondrial morphology, it is currently debated whether alteration of mitochondrial morphology is per se responsible for apoptosis execution or prevention. This study was undertaken to examine the effect of histone deacetylase (HDAC) inhibitors on mitochondrial fusion-fission equilibrium. The mechanism underlying HDAC inhibitor-induced alteration of mitochondrial morphology was examined in various cells including primary cultured cells and untransformed and cancer cell lines treated with seven different HDAC inhibitors. Suberoylanilide hydroxamic acid (SAHA)-induced mitochondrial elongation in both Hep3B and Bcl-2-overexpressing Hep3B cells, apart from its apoptosis induction function. SAHA significantly decreased the expression of mitochondrial fission protein Fis1 and reduced the translocation of Drp1 to the mitochondria. Fis1 overexpression attenuated SAHA-induced mitochondrial elongation. In addition, depletion of mitochondrial fusion proteins, Mfn1 or Opa1, by RNA interference also attenuated SAHA-induced mitochondrial elongation. All of the HDAC inhibitors we examined induced mitochondrial elongation in all the cell types tested at both subtoxic and toxic concentrations. These results indicate that HDAC inhibitors induce mitochondrial elongation, irrespective of the induction of apoptosis, which may be linked to alterations of mitochondrial dynamics regulated by mitochondrial morphology-regulating proteins. Since mitochondria have recently emerged as attractive targets for cancer therapy, our findings that HDAC inhibitors altered mitochondrial morphology may support the rationale for these agents as novel therapeutic approaches against cancer. Further, the present study may provide insight into a valuable experimental strategy for simple manipulation of mitochondrial morphology.     

Selenium-containing analogs of SAHA induce cytotoxicity in lung cancer cells

Cancer therapy has moved beyond conventional chemotherapeutics to more mechanism-based targeted approaches. Studies demonstrate that histone deacetylase (HDAC) is a promising target for anticancer agents. Numerous, structurally diverse, hydroxamic acid derivative, HDAC inhibitors have been reported and have been shown to induce growth arrest, differentiation, autophagy, and/or apoptotic cell death by inhibiting multiple signaling pathways in cancer cells. Suberoylanilide hydroxamic acid (SAHA) has emerged as an effective anticancer therapeutic agent and was recently approved by the FDA for the treatment of advanced cutaneous T-cell lymphoma. In our previous study, we reported the development of the novel, potent, selenium-containing HDAC inhibitors (SelSA-1 and SelSA-2). In this study, the effects of SelSA-1 and SelSA-2 on signaling pathways and cytotoxicity were compared with the known HDAC inhibitor, SAHA, in lung cancer cell lines. After 24 h of treatment, SelSA-1 and SelSA-2 inhibited lung cancer cell growth to a greater extent than SAHA in a dose-dependent manner with IC₅₀ values at low micromolar concentrations. SelSA-1 and SelSA-2 inhibited ERK and PI3K-AKT signaling pathways while simultaneously increasing in autophagy in A549 cells in a time dependent manner. This preliminary study demonstrates the effectiveness of the selenium-containing analogs of SAHA, SelSA-1, and SelSA-2, as HDAC inhibitors and provides insight into the improvement and/or development of these analogs as a therapeutic approach for the treatment of lung cancer.     

HDAC inhibitors induce apoptosis in glucocorticoid-resistant acute lymphatic leukemia cells despite a switch from the extrinsic to the intrinsic death pathway

Inhibitors of histone deacetylases (HDACi's) are promising novel tools for cancer therapy. We have compared the growth inhibitory and apoptogenic potential of the pan-HDACi SAHA and the sub-class I selective HDAC inhibitor MS275, as well as valproic acid (VPA) on glucocorticoid sensitive and resistant B (B-ALL) and T (T-ALL) cell acute lymphoblastic leukemia cells and patients blasts. In contrast, to our previous results with U937 acute myeloid leukemia (AML) cells which showed a similar activity of MS275 and SAHA in growth inhibition and apoptosis induction, both B and T-ALL cells were much more efficiently killed by SAHA and VPA than by MS275. The same relative potency was observed with some patient ALL blasts treated ex vivo. SAHA displayed similar efficacy on glucocorticoid-sensitive and insensitive ALL cells but did not synergize with dexamethasone. In studying mediators of apoptosis we found that the TRAIL receptor DR5 is constitutively expressed in glucocorticoid-sensitive CEM-C7 cells which are also TRAIL sensitive. In contrast, glucocorticoid-insensitive CEM-C1 cells do not express DR5 and are insensitive to TRAIL. However, SAHA induces, in addition to p21(WAF1/CIP1) also re-expression of DR5. Importantly, SAHA-induced apoptosis of CEM-C7 cells operates through initiator caspase 10, while it induces apoptosis of CEM-C1 cells through the intrinsic, as well as through caspase-independent death pathways. Our data suggest that the generation of resistance to glucocorticoids has dramatically altered death signaling in these cells and that SAHA overcomes these restrictions by inducing alternative death pathways.     

Suberoylanilide hydroxamic acid induces ROS-mediated cleavage of HSP90 in leukemia cells

Heat shock protein 90 (HSP90) is a molecular chaperone that supports stability of client proteins. We found that HSP90 was cleaved to 55 kDa protein after treatment with histone deacetylase (HDAC) inhibitors including suberoylanilide hydroxamic acid (SAHA) in several leukemia cell lines. We further analyzed molecular changes induced by SAHA in K562 cells. The SAHA-induced cleavage of HSP90 was blocked by a pan-caspase inhibitor, z-VAD-fmk, implying that the process is dependent on caspase activity. However, the experiments using antagonistic and agonistic Fas antibodies revealed that the cleavage of HSP90 was not dependent on Fas signaling. SAHA induced generation of reactive oxygen species (ROS), and the cleavage of HSP90 was blocked by a ROS scavenger N-acetylcystein (NAC). We also confirmed that hydrogen peroxide (H₂O₂) induced cleavage of HSP90 in a similar manner. Caspase 2, 3, 4, 6, 8, and 10 were activated by treatment with SAHA, and the activities were reduced by the pretreatment of NAC. Treatment of the cells with caspase 10 inhihitor, but not other inhibitors of caspases activated by SAHA, prevented cleavage of HSP90 by SAHA. SAHA-induced ROS generation and HSP90 cleavage were dependent on newly synthesized unknown proteins. Taken together, our results suggest that the cleavage of HSP90 by SAHA is mediated by ROS generation and caspase 10 activation. HSP90 cleavage may provide an additional mechanism involved in anti-cancer effects of HDAC inhibitors.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0533-4) contains supplementary material, which is available to authorized users.     

c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.     

Suberoylanilide Hydroxamic Acid Triggers Autophagy by Influencing the mTOR Pathway in the Spinal Dorsal Horn in a Rat Neuropathic Pain Model

Histone acetylation levels can be upregulated by treating cells with histone deacetylase inhibitors (HDACIs), which can induce autophagy. Autophagy flux in the spinal cord of rats following the left fifth lumber spinal nerve ligation (SNL) is involved in the progression of neuropathic pain. Suberoylanilide hydroxamic acid (SAHA), one of the HDACIs can interfere with the epigenetic process of histone acetylation, which has been shown to ease neuropathic pain. Recent research suggest that SAHA can stimulate autophagy via the mammalian target of rapamycin (mTOR) pathway in some types of cancer cells. However, little is known about the role of SAHA and autophagy in neuropathic pain after nerve injury. In the present study, we aim to investigate autophagy flux and the role of the mTOR pathway on spinal cells autophagy activation in neuropathic pain induced by SNL in rats that received SAHA treatment. Autophagy-related proteins and mTOR or its active form were assessed by using western blot, immunohistochemistry, double immunofluorescence staining and transmission electron microscopy (TEM). We found that SAHA decreased the paw mechanical withdrawal threshold (PMWT) of the lower compared with SNL. Autophagy flux was mainly disrupted in the astrocytes and neuronal cells of the spinal cord dorsal horn on postsurgical day 28 and was reversed by daily intrathecal injection of SAHA (n?=?100 nmol/day or n?=?200 nmol/day). SAHA also decreased mTOR and phosphorylated mTOR (p-mTOR) expression, especially p-mTOR expression in astrocytes and neuronal cells of the spinal dorsal horn. These results suggest that SAHA attenuates neuropathic pain and contributes to autophagy flux in astrocytes and neuronal cells of the spinal dorsal horn via the mTOR signaling pathway.

     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

Suberoylanilide Hydroxamic Acid Induces Hypersensitivity to Radiation Therapy in Acute Myelogenous Leukemia Cells Expressing Constitutively Active FLT3 Mutants

Histone deacetylase inhibitors (HDIs) have shown promise as candidate radiosensitizer for many types of cancers. However, the mechanisms of action are not well understood, and whether they could have clinical impact on radiotherapy for leukemia is unclear. In this study, we demonstrate that suberoylanilide hydroxamic acid (SAHA) can increase radiosensitivity of acute myeloid leukemia (AML) cells through posttranslational modification of Rad51 protein responses and selective inhibition of the homology-directed repair (HDR) pathway. Our data also showed that AML cells with mutant, constitutively active FMS-like tyrosine kinase-3 (FLT3) were more radiation sensitive, caused by compromised non-homologous end joining (NHEJ) repair. Furthermore, SAHA-induced radiosensitization were enhanced in AML cells with expression of these FLT3 mutants. The results of this study suggest that SAHA, a recently approved HDI in clinical trials, may act as a candidate component for novel conditioning regimens to improve efficacy for AML patients undergoing radiotherapy and chemotherapy.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

Modulating autophagy for therapeutic benefit

Autophagy is an ancient cell survival pathway that allows cells to recoup ATP and essential building blocks for biosynthesis when they are starved of nutrients or when they are exposed to hypoxia, which are hallmarks of the tumor microenvironment. This pathway involves the formation of double-membraned vesicles, coined autophagosomes, which envelop bulk cellular material and/or organelles and that subsequently fuse with lysosomes that degrade their cargo. Autophagy has been suggested to play important roles in chemoresistance of cancer to some therapeutic agents, which typically induce an apoptotic response. For example, the histone deacetylase inhibitor SAHA induces both apoptosis and autophagy, suggesting that agents that disrupt the autophagy pathway might augment its efficacy as a therapeutic agent. We tested this notion in a model of Imatinib-refractory chronic myelogenous leukemia (CML) and in imatinib-resistant primary CML cells from patients bearing mutations in Bcr-Abl, including the T315I mutation that causes resistance to currently utilized tyrosine kinase inhibitors and translates into a very poor clinical prognosis. Agents that disrupt autophagy were shown to synergize with SAHA in provoking apoptotic death of these refractory tumors. These findings support the use of agents that disrupt the autophagy pathway in settings of chemorefractory malignancies.