mTOR Inhibitors and its Role in the Treatment of Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinomas (HNSCC) represent 6% of all cancers diagnosed each year in the United States, affecting approximately 43,000 new patients and resulting in approximately 12,000 deaths. Currently, three main rapalogs exist for the treatment of cancer: CCI-779 (temsirolimus), RAD001 (everolimus), and AP235373 (deforolimus). Clinicians managing HNSCC need to be aware of the three rapalogs. Extensive evidence has shown rapamycin-analogs to be effective agents in the treatment of a number of solid tumors. While extensive preclinical data suggests that HNSCC would be an appropriate tumor type to benefit from inhibition of the mTOR pathway, limited clinical data is yet available to support this. Numerous phase II trials evaluating mTOR inhibitors for use in HNSCC are currently recruiting patients.     

Individualizing antimetabolic treatment strategies for head and neck squamous cell carcinoma based on TP53 mutational status

BACKGROUND:

Mutations in the tumor protein 53 (TP53) tumor suppressor gene are common in head and neck squamous cell carcinoma (HNSCC) and correlate with radioresistance. Currently, there are no clinically available therapeutic approaches targeting p53 in HNSCC. In this report, the authors propose a strategy that uses TP53 mutational status to individualize antimetabolic strategies for the potentiation of radiation toxicity in HNSCC cells.

METHODS:

Glycolytic flux and mitochondrial respiration were evaluated in wild‐type (wt) and mutant (mut) TP53 HNSCC cell lines. Sensitivity to external‐beam radiation (XRT) was measured using a clonogenic assay.

RESULTS:

HNSCC cells that expressed mutTP53 demonstrated radioresistance compared with HNSCC cells that expressed wtTP53. Glycolytic inhibition potentiated radiation toxicity in mutTP53‐expressing, but not wtTP53‐expressing, HNSCC cells. The relative sensitivity of mutTP53 HNSCC cells to glycolytic inhibition was caused by a glycolytic dependence associated with decreased mitochondrial complex II and IV activity. The wtTP53‐expressing cells maintained mitochondrial reserves and were relatively insensitive to glycolytic inhibition. Inhibition of respiration using metformin increased glycolytic dependence in wtTP53‐expressing cells and potentiated the effects of glycolyic inhibition on radiation toxicity.

CONCLUSIONS:

TP53 mutation in HNSCC cells was correlated with a metabolic shift away from mitochondrial respiration toward glycolysis, resulting in increased sensitivity to the potentiating effects of glycolytic inhibition on radiation toxicity. In contrast, wtTP53‐expressing cells required inhibition of both mitochondrial respiration and glycolysis to become sensitized to radiation. Therefore, the authors concluded that TP53 mutational status may be used as a marker of altered tumor cell metabolism to individualize HNSCC treatment selection of specific, targeted metabolic agents that can overcome cellular resistance to radiation therapy. Cancer 2012;. © 2011 American Cancer Society.     

Mammalian target of rapamycin inhibitors as possible adjuvant therapy for microscopic residual disease in head and neck squamous cell cancer

Molecular therapeutics identifies an aberration in tumors to select patients that benefit from molecular targeted therapy. Overexpression of eIF4E in histologically "tumor-free" surgical margins of head and neck squamous cell cancer (HNSCC) patients is an independent predictor of recurrence and is functionally activated through the Akt/mammalian target of rapamycin (mTOR) pathway. Although mTOR inhibitors are cytostatic agents, best used in combination therapy, we hypothesize that they can be used as long-term single agents in an HNSCC model of minimal residual disease (MRD). CCI-779, an mTOR inhibitor, arrested growth of a phosphatase and tensin homologue deleted on chromosome 10 (PTEN) abnormal HNSCC cell line FaDu, inhibiting phosphorylation of 4E-binding protein 1, resulting in increased association with eIF4E and inhibition of basic fibroblast growth factor and vascular endothelial growth factor. Fluorescence in situ hybridization detected PTEN abnormalities in 68% of patient tumors and 35% of tumor-free margins. CCI-779 inhibited growth of established tumors in nude mice. However, in the MRD model, there were significant differences in the tumor-free rate between the control (4%) and the treatment group (50%), and the median tumor-free time was 7 versus 18 days, respectively (P < 0.0001). In those animals that formed tumors, CCI-779 caused a significant decrease in the tumor volume. The Kaplan-Meier curve showed that CCI-779 significantly increased survival (P < 0.0001). The mTOR pathway was inhibited in peripheral blood mononuclear cells potential surrogate markers of response to therapy. Stable transfection of FaDu with luciferase allowed us to monitor the effects of CCI-779 with bioluminescence imaging in the MRD model. These results pave the way for a clinical trial using targeted molecular therapy with CCI-779 as a single agent for mTOR-activated residual cells.     

The autophagy associated gene,ULK1, promotes tolerance to chronic and acute hypoxia

Background and purpose

Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment.

Material and methods

Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients.

Results

ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia.

Conclusions

We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.     

Molecular and cytogenetic subgroups of oropharyngeal squamous cell carcinoma.

PURPOSE: The aim of this study was to acquire further insights into the pathogenetic pathways of head and neck squamous cell carcinomas (HNSCC) that may be useful for identifying new biomarkers instrumental in developing more specific treatment approaches. EXPERIMENTAL DESIGN: Cell cycle regulators and epidermal growth factor receptor (EGFR) and BRAF genes were analyzed in a series of 90 oropharyngeal SCCs of a cohort of surgically treated patients from a single institution, and the results were matched with the presence of high-risk human papillomavirus (HR-HPV) DNA and the TP53 status. RESULTS: At least four distinct groups of tumors were identified sharing a common histology but displaying different molecular/cytogenetic patterns: (a) 19% were HPV-positive SCCs whose lack of alterations of the investigated genes could explain their particular natural history, which requires less aggressive treatment; (b) 37% were HPV-negative SCCs carrying TP53 mutations, which may be more effectively treated by drugs acting through p53-independent apoptosis; (c) 34% were HPV-negative SCCs carrying wild-type TP53 and loss of 9p21 (p16INK4a and p15INK4b) and/or cyclin D1 overexpression that justify treatment with DNA-damaging drugs followed by cell cycle inhibitors; and (d) 10% were HPV-negative lacking tumor suppressor genes and cell cycle alterations. The second, third, and fourth groups also showed an increased copy number of EGFR and chromosome 7 (43%) that might justify the additional or alternative use of EGFR inhibitors. CONCLUSIONS: Our findings suggest that assessing HPV, TP53, 9p21, and EGFR status may be crucial to finding more tailored and beneficial treatments for oropharyngeal SCCs.     

Mutation of the TP53 gene in acute lymphoblastic leukemia does not affect survival outcomes after haploidentical hematopoietic stem cell transplantation

Previous studies have demonstrated that TP53 mutation is correlated with insufficient therapy response and unfavorable prognosis in acute lymphoblastic leukemia (ALL). Few studies have investigated the impact of TP53 mutation in ALL patients after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). We completed a retrospective study of 65 ALL patients with available TP53 status who underwent haplo-HSCT. They were divided into a TP53 mutation group (TP53^mut) and a TP53 wild-type (TP53^wt) group. TP53^mut showed comparable 2-year cumulative incidence of relapse (CIR) rates (13.1% vs 12.5%, P = .96) and 2-year leukemia-free survival (LFS) (74.2% vs 77.4%, P = .80) with TP53^wt. No significant differences in 2-year overall survival (OS) rates (82.9% vs 87.3%, P = .61) or 2-year NRM rates (12.7% vs 10.2%, P = .69) were observed in TP53^mut and TP53^wt patients. Multivariate analysis suggested that white blood cell (WBC) count at initial diagnosis (>50 × 10⁹/L: hazard ratio [HR] = 3.860, P = .016) and age (>40 years old: HR = 4.120, P = .012) are independent risk factors for 2-year LFS. Our study showed that TP53 mutations may not be related to the unfavorable impact on survival in ALL patients after treatment with haplo-HSCT. The present results suggested that haplo-HSCT may eliminate the poor prognosis effect of TP53 mutation in ALL.     

熊果酸对胃癌细胞株MGC-803 凋亡和自噬的调控及其作用机制

[摘要] 目的:观察熊果酸(UA)对胃癌细胞株MGC-803 自噬和凋亡的调控作用,并探讨UA基于PI3K/AKT/mTOR信号通路诱发MGC-803 细胞自噬的机制。方法:体外培养人胃癌细胞株MGC-803,分为空白对照组、UA干预组和UA+3-MA组。采用流式细胞术检测各组细胞凋亡水平,双荧光mRFP-eGFP-LC3 质粒转染细胞法检测各组细胞自噬发生情况,qPCR实验检测各组细胞LC3B、BAX、Bcl-2 mRNA的表达水平,WB实验检测各组细胞Ⅰ型PI3K、p-AKT、p-mTOR、ULK1、LC3B、BAX、Bcl-2 蛋白的表达水平。结果: 与空白对照组相比,UA干预组细胞凋亡率显著增加（P<0.05）;与UA干预组相比,UA+3-MA组细胞凋亡率显著降低（P<0.05）。双荧光mRFP-eGFP-LC3 质粒转染法显示,与空白对照组相比,UA 干预组绿色和红色荧光亮点数均显著增加（P<0.05）;与UA干预组相比,UA+3-MA组绿色和红色荧光亮点数均显著减少（P<0.05）。qPCR和WB实验结果显示,与空白对照组相比,UA干预组BAX和LC3B mRNA和蛋白以及ULK1 蛋白表达水平均显著上调,Bcl-2 基因和蛋白表达水平以及Ⅰ型PI3K、p-AKT、p-mTOR蛋白表达水平均显著下调（均P<0.05）;与UA干预组相比,UA+3-MA组BAX、LC3B mRNA和蛋白表达水平显著下调,Bcl-2 基因和蛋白表达水平显著上调（均P<0.05）,Ⅰ型PI3K、p-AKT、p-mTOR 和ULK1 蛋白表达水平无显著差异（P>0.05）。结论: UA诱导的自噬可以促进胃癌细胞MGC-803 的凋亡,其机制可能与UA参与调控PI3K/AKT/mTOR信号通路相关蛋白表达有关。     

Inhibition of mammalian target of rapamycin signaling potentiates the effects of all‐trans retinoic acid to induce growth arrest and differentiation of human acute myelogenous leukemia cells

Our study explored the drug interaction of all‐trans retinoic acid (ATRA) and RAD001 (everolimus), the inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in acute myelogenous leukemia (AML) NB4 and HL60 cells. RAD001 (10 nM) significantly enhanced the ATRA‐induced growth arrest and differentiation of these cells, as measured by colony‐forming assay and cell cycle analysis, and expression of CD11b cell surface antigen and nitroblue tetrazolium reduction, respectively. ATRA (0.1–1 μM) upregulated levels of RTP801, a negative regulator of mTORC1, and inhibited mTORC1 signaling as assessed by measurement of the levels of p‐p70S6K and p‐4E‐BP1 in HL60 and NB4 cells. ATRA (0.1–1 μM) in combination with RAD001 (10 nM) strikingly downregulated the levels of p‐70S6K and p‐4E‐BP1 without affecting the total amount of these proteins. Notably, RAD001 (10 nM) significantly augmented ATRA‐induced expression of CCAAT/enhancer‐binding protein ε (C/EBPε) and p27^kip1 and downregulated levels of c‐Myc in these cells. Furthermore, RAD001 (5 mg/kg) enhanced the ability of ATRA (10 mg/kg) to inhibit the proliferation of HL60 cells growing as tumor xenografts in immune‐deficient nude mice. Taken together, concomitant blockade of the RA and mTORC1 signaling may be a promising treatment strategy for individuals with AML. © 2009 UICC     

GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth

The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin''s growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.     

自噬在视网膜母细胞瘤Y79细胞顺铂耐药中的作用及其机制

目的 研究自噬在视网膜母细胞瘤Y79细胞顺铂耐药中的作用及其机制。方法 CCK-8法检测细胞IC50;将Y79细胞随机分为对照组（Control）、顺铂组（Cis）和顺铂联合应用自噬阻断剂3-甲基腺嘌呤组（Cis+3-MA）,Western blot法、细胞自噬染色检测试剂盒（MDC法）和透射电子显微镜观察细胞自噬情况;CCK-8法检测顺铂对细胞的抑制率变化,Annexin V/PI双染流式法检测细胞凋亡变化,q-PCR检测相关耐药基因转录水平,Fluo-4 AM钙离子荧光探针染色检测细胞内钙离子变化,Western blot法检测CaMKK2、p-AMPK、mTORC1、LC3Ⅱ的表达。结果 视网膜母细胞瘤Y79细胞在顺铂的诱导下发生自噬,加入自噬阻断剂3-MA后细胞自噬水平下调。与顺铂组相比,Cis+3-MA组顺铂抑制率增加,凋亡率增加,相关耐药基因转录水平下调。细胞加入顺铂后,细胞内钙离子水平增加,CaMKK2、p-AMPK、LC3Ⅱ表达上调,mTORC1表达下调。结论 顺铂诱导肿瘤细胞产生的自噬对视网膜母细胞瘤细胞Y79耐药起保护作用,抑制自噬可以改善肿瘤细胞对顺铂的耐药性。顺铂可能是通过Ca2+激活CaMKK2/AMPK/mTORC1通路诱导Y79细胞自噬。     

Combinations of mTORC1 inhibitor RAD001 with gemcitabine and paclitaxel for treating non-Hodgkin lymphoma

Single-agent mammalian target of rapamycin complex 1 (mTORC1) inhibitors have recently been reported as effective salvage treatment in non-Hodgkin lymphoma (NHL). The combined effect of mTORC1 inhibitor, RAD001, with chemotherapeutic agents used for relapsed or refractory NHL was examined. Synergistic interactions were observed for RAD001 plus gemcitabine or paclitaxel in six NHL cell lines; enhanced gemcitabine- and paclitaxel-induced caspase-dependent apoptosis associated with down-regulation of mTOR signaling was detected. Synergistic interactions were also observed with RAD001 plus gemcitabine and paclitaxel. In conclusion, synergistic cytotoxicity was observed with RAD001 plus gemcitabine and paclitaxel in NHL cells. Combination therapy with these three drugs should be examined in patients with refractory or relapsed NHL.     

p53-Reactivating small molecules induce apoptosis and enhance chemotherapeutic cytotoxicity in head and neck squamous cell carcinoma

We evaluate whether p53-reactivating (p53RA) small molecules induce p53-dependent apoptosis in head and neck squamous cell carcinoma (HNSCC), a question that has not been previously addressed in head and neck cancer. PRIMA-1, CP-31398, RITA, and nutlin-3 were tested in four human HNSCC cell lines differing in TP53 status. Cell growth, viability, cell cycle progression, and apoptosis after treatment with p53RA small molecules individually or in combination with chemotherapeutic agents were assessed. Prominent p53 reactivation was observed in mutant TP53-bearing tumor cell lines treated with PRIMA-1 or CP-31398, and in wild-type TP53-bearing cell lines treated with nutlin-3. Cell-cycle arrest and apoptosis induced by p53RA small molecules were associated with upregulation of p21 and BAX, and cleavage of caspase-3. Nutlin-3 showed maximal growth suppression in tumor cells showing MDM2-dependent p53 degradation. High-dose treatment with p53RA small molecules also induced apoptosis in cell lines independent of p53 or MDM2 expression. In combination therapy, p53RA small molecules enhanced the anti-tumor activity of cisplatin, 5-fluorouracil, paclitaxel, and erlotinib against HNSCC cells. The p53RA small molecules effectively restored p53 tumor-suppressive function in HNSCCs with mutant or wild-type TP53. The p53RA agents may be clinically useful against HNSCC, in combination with chemotherapeutic drugs.     

Generation and molecular characterization of head and neck squamous cell lines of fanconi anemia patients

Patients with Fanconi anemia (FA) are prone to develop malignancies at an early age. Besides hematologic malignancies, squamous cell carcinomas in the anogenital region and head and neck are also frequently found in these patients. The aim of this study was to generate a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts of FA HNSCC, and to characterize these cell lines in comparison with a panel of seven cell lines from patients with sporadic HNSCC. Analyses have been done on sensitivity to DNA cross-linking agents, loss of heterozygosity profile, TP53 mutations, TP53 polymorphisms and the presence of human papillomavirus. Four FA HNSCC cell lines were established. Sensitivity to DNA cross-linking agents (cisplatin) in the FA HNSCC cell lines was on average 10 times higher as compared with the sporadic HNSCC cell lines. Human papillomavirus was not detected in any of the FA or sporadic cell lines. No differences were found in loss of heterozygosity pattern, TP53 mutation frequency and TP53 polymorphism between FA and sporadic HNSCC cell lines. This is the first report on the generation of squamous cell lines of FA patients. The FA HNSCC cell lines we have generated may be utilized for future studies and might aid in the development of new preventive therapies for FA patients. The genetic characteristics of these cell lines suggest that FA HNSCC are not very different from sporadic HNSCC, except for the sensitivity to cisplatin which is consistent with the known cellular FA phenotype.     

Patterns of antibody responses to nonviral cancer antigens in head and neck squamous cell carcinoma patients differ by human papillomavirus status

There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤ 10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC.     

三七皂苷R1调控PI3K/AKT/mTOR信号通路诱导人下咽鳞状细胞癌FaDu细胞凋亡和自噬北大核心

目的:探讨三七皂苷R1(notoginsenoside R1,NGR1)对人下咽鳞状细胞癌(hypopharyngeal squamous cell carcinoma,HSCC)FaDu细胞凋亡以及自噬的影响,并对其涉及的信号通路进行研究。方法:75μmol/L、150μmol/L、300μmol/L NGR1作用于FaDu细胞24 h后,采用MTT检测细胞增殖能力;流式细胞术检测细胞凋亡;自噬双标腺病毒检测自噬流;Western blot检测自噬相关蛋白LC3Ⅱ/LC3Ⅰ以及PI3K/AKT/mTOR信号通路相关蛋白表达水平。结果:NGR1能够抑制FaDu细胞的增殖并促进细胞凋亡;NGR1可诱导FaDu细胞自噬,并呈一定浓度依赖性;Western blot结果显示,NGR1作用于Fa Du细胞24 h后,LC3Ⅱ表达明显增加,而p-PI3K、p-AKT、p-m TOR表达相较于Control组明显下降。结论:NGR1可抑制Fa Du细胞增殖,诱导细胞凋亡与自噬,其机制可能与抑制PI3K/AKT/m TOR信号通路有关。     

Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing

BackgroundTo determine genomic alterations in head and neck squamous cell carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained through routine clinical practice, selected cancer-related genes were evaluated and compared with alterations seen in frozen tumors obtained through research studies.Patients and methodsDNA samples obtained from 252 FFPE HNSCC were analyzed using next-generation sequencing-based (NGS) clinical assay to determine sequence and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV in situ hybridization (ISH) and p16 immunohistochemical (IHC) staining in a subset of tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and University of Chicago public datasets were analyzed for comparison.ResultsAmong 252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete concordance with NGS-derived HPV status. The most common genes with genomic alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were frequently altered. The HPV-positive oropharynx and HPV-positive nasal cavity/paranasal sinus carcinoma shared similar mutational profiles.ConclusionThe genomic profile of FFPE HNSCC tumors obtained through routine clinical practice is comparable with frozen tumors studied in research setting, demonstrating the feasibility of comprehensive genomic profiling in a clinical setting. However, the clinical significance of these genomic alterations requires further investigation through application of these genomic profiles as integral biomarkers in clinical trials.     

Multiple mechanisms contribute to the synergistic anti-myeloma activity of the pan-histone deacetylase inhibitor LBH589 and the rapalog RAD001

We examined the pre-clinical activity of pan-histone deacetylase inhibitor LBH589 in combination with mTORC1 inhibitor RAD001 and observed that the drug combination strongly synergized in inducing cytotoxicity in multiple myeloma (MM) cells. LBH589 caused an increase in acetylated histones and RAD001 inhibited mTORC1 activity. RAD001 caused potent G0/G1 arrest while LBH589 induced pronounced apoptosis, both of which were enhanced when the drugs were used in combination. LBH589/RAD001 combination led to down regulation of pStat3, cyclins, CDKs and XIAP and up regulation of pro-apoptotic Bcl-2 family proteins. A clinical trial is underway using LBH589/RAD001 combination in relapsed MM patients.