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PDF file, 871K, S1 Chromogenic staining (DAB, brown) of paraffin-embedded DLBCL cell lines DHL4, and Toledo, untreated and BCR crosslinked, with anti-pLYN (Y396), anti-pSYK (Y323), and anti-pBTK (Y551); S2 Schematic illustration of Tissuequest analysis workflow. Individual cells are first identified by finding the nuclei (DAPI); S3 Qualitative Western blot of whole cell lysates for untreated (BCR-) and crosslinked (BCR+) cell lines; S4 S4 Chromogenic staining (DAB, brown) of two paraffin-embedded patient DLBCL specimens with anti-pLYN (Y396), anti-pSYK (Y323), and anti-pBTK (Y551); S5 (A) Schematic illustration of Tissuequest workflow extended to subcellular localization of FOXO1. In this example there are 83% of all cells that are positive for FOXO1 and 67% of the cells show cytoplasmic FOXO1 staining (67%). The ratio of these percentages is used to calculate the Fcyt score; S6 (A) Chromogenic staining (DAB, brown) of paraffin-embedded DLBCL cell lines DHL4, and Toledo, untreated and BCR crosslinked, with anti- FOXO1; S7 (A) IHC staining of paraffin-embedded DLBCL cell lines U2932, DHL6 and Toledo, untreated and crosslinked, with anti-pAKTsub (red) and DAPI (blue); S8 (A) Comparison of immunofluorescent images evaluated by an expert using a 3-tier system (0 - negative, 1 - weak positive, 2 - strong positive); S9 Comparison of FOXO1 cytoplasmic localization and BCR signaling in primary DLBCL tumors.

PDF file - 462K, Table S1: pLYN, pSYK and pBTK levels in DLBCL cell lines; S2 DLBCL cell line classification

Purpose: B-cell receptor (BCR)–mediated signaling is important in the pathogenesis of a subset of diffuse large B-cell lymphomas (DLBCL) and the BCR-associated kinases SYK and BTK have recently emerged as potential therapeutic targets. We sought to identify a signature of activated BCR signaling in DLBCL to aid the identification of tumors that may be most likely to respond to BCR-pathway inhibition.Experimental Design: We applied quantitative immunofluorescence (qIF) using antibodies to phosphorylated forms of proximal BCR signaling kinases LYN, SYK, and BTK and antibody to BCR-associated transcription factor FOXO1 on BCR-cross-linked formalin-fixed paraffin-embedded (FFPE) DLBCL cell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n = 154).Results: A robust signature of active BCR signaling was identified and validated in BCR-cross-linked DLBCL cell lines and in 71/154 (46%) of the primary DLBCL patient specimens. Further analysis of the primary biopsy samples revealed increased nuclear exclusion of FOXO1 among DLBCL with qIF evidence of active BCR signaling compared with those without (P = 0.004). Nuclear exclusion of FOXO1 was also detected in a subset of DLBCL without evidence of proximal BCR signaling suggesting that alternative mechanisms for PI3K/AKT activation may mediate FOXO1 subcellular localization in these cases.Conclusion: This study establishes the feasibility of detecting BCR activation in primary FFPE biopsy specimens of DLBCL. It lays a foundation for future dissection of signal transduction networks in DLBCL and provides a potential platform for evaluating individual tumors in patients receiving novel therapies targeting the BCR pathway. Clin Cancer Res; 18(22); 6122–35. ©2012 AACR.

Novel drug targets for sustained reduction in body mass index (BMI) are needed to curb the epidemic of obesity, which affects 650 million individuals worldwide and is a causal driver of cardiovascular and metabolic disease and mortality. Previous studies reported that the Arg95Ter nonsense variant of GPR151, an orphan G protein-coupled receptor, is associated with reduced BMI and reduced risk of Type 2 Diabetes (T2D). Here, we follow up on GPR151 with the Pakistan Genome Resource (PGR), which is one of the largest exome biobanks of human homozygous loss-of-function carriers (knockouts) in the world. Among PGR participants, we identify 3 GPR151 putative loss-of-function (plof) variants (Arg95Ter, Tyr99Ter, and Phe175LeufsTer7) with a cumulative allele frequency of 2.2% and present at homozygosity. We confirm these alleles in vitro as loss-of-function. We test if GPR151 plof is associated with BMI, T2D, or other metabolic traits. GPR151 deficiency in complete human knockouts is not associated with a clinically significant difference in BMI. Moreover, loss of GPR151 confers a nominally significant increase in risk of T2D (odds ratio = 1.2, p value = 0.03). Relative to wild-type mice, Gpr151-/- animals exhibit no difference in body weight on normal chow, and higher body weight on a high-fat diet, consistent with the findings in humans. Together, our findings indicate that GPR151 antagonism is not a compelling therapeutic approach for obesity.

The serine protease Factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anti-coagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement Factor D inhibitor and exhibited sub-micromolar FXIa activity and an encouraging ADME profile while being devoid of peptidomimetic architecture. Optimization of interactions in the S1, S1β, and S1` pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with sub-nanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a pre-clinical PK profile consistent with bid dosing in patients.

Purpose: B-cell receptor (BCR)–mediated signaling is important in the pathogenesis of a subset of diffuse large B-cell lymphomas (DLBCL) and the BCR-associated kinases SYK and BTK have recently emerged as potential therapeutic targets. We sought to identify a signature of activated BCR signaling in DLBCL to aid the identification of tumors that may be most likely to respond to BCR-pathway inhibition. Experimental Design: We applied quantitative immunofluorescence (qIF) using antibodies to phosphorylated forms of proximal BCR signaling kinases LYN, SYK, and BTK and antibody to BCR-associated transcription factor FOXO1 on BCR-cross-linked formalin-fixed paraffin-embedded (FFPE) DLBCL cell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n = 154). Results: A robust signature of active BCR signaling was identified and validated in BCR-cross-linked DLBCL cell lines and in 71/154 (46%) of the primary DLBCL patient specimens. Further analysis of the primary biopsy samples revealed increased nuclear exclusion of FOXO1 among DLBCL with qIF evidence of active BCR signaling compared with those without (P = 0.004). Nuclear exclusion of FOXO1 was also detected in a subset of DLBCL without evidence of proximal BCR signaling suggesting that alternative mechanisms for PI3K/AKT activation may mediate FOXO1 subcellular localization in these cases. Conclusion: This study establishes the feasibility of detecting BCR activation in primary FFPE biopsy specimens of DLBCL. It lays a foundation for future dissection of signal transduction networks in DLBCL and provides a potential platform for evaluating individual tumors in patients receiving novel therapies targeting the BCR pathway. Clin Cancer Res; 18(22); 6122–35. ©2012 AACR.

MSI2 is highly expressed in human myeloid leukemia (AML) cell lines, and high expression of MSI2 mRNA is associated with decreased survival in AML, suggesting its use as a new prognostic marker. To test this, we measured MSI2 protein level by immunohistochemistry in 120 AML patients. Most cases (70%) showed some nuclear or cytoplasmic positivity, but the percentage of positive cells was low in most cases. Despite this, MSI2 protein expression was negatively associated with outcome, particularly for patients with good cytogenetic subgroup. For practical diagnostic purposes, the strongest significance of association was seen in cases with > 1% of cells showing strong MSI2 staining, these having a very poor outcome (P < .0001). Multivariate analysis with cytogenetic category, age, white cell count, and French-American-British subtype demonstrated that nuclear MSI2 levels were independently predictive of outcome (P = .0497). These results confirm the association of MSI2 expression with outcome in AML at the protein level and demonstrate the utility of MSI2 protein as a clinical prognostic biomarker. In addition, although positive at some level in most cases, its prognostic power derived from few positive cells, supporting its role in control of normal hematopoietic stem cell function and highlighting its role in disease progression.

Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBVdriven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid Bcell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydratebinding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4 Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)–mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8 T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors. Fil: Ouyang, Ying. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Juszczynski, Przemyslaw. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Rodig, Scott J.. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Green, Michael R.. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: O´ Donnell, Evan. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Currie, Treeve. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Armant, Myriam. Immune Disease Institute/Harvard Medical School. Center for Human Cell Therapy; Estados Unidos Fil: Takeyama, Kunihiko. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Monti, Stefano. Broad Institute; Estados Unidos Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Ritz, Jerome. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Kutok, Jeffery L.. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Shipp, Margaret A.. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q− syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q− syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q− syndrome, DBA, and perhaps other bone marrow failure syndromes.

Classical Hodgkin lymphoma (cHL) and mediastinal large B-cell lymphoma (MLBCL) are lymphoid malignancies with certain shared clinical, histologic, and molecular features. Primary cHLs and MLBCLs include variable numbers of malignant cells within an inflammatory infiltrate, suggesting that these tumors escape immune surveillance. Herein, we integrate high-resolution copy number data with transcriptional profiles and identify the immunoregulatory genes, PD-L1 and PD-L2, as key targets at the 9p24.1 amplification peak in HL and MLBCL cell lines. We extend these findings to laser-capture microdissected primary Hodgkin Reed-Sternberg cells and primary MLBCLs and find that programmed cell death-1 (PD-1) ligand/9p24.1 amplification is restricted to nodular sclerosing HL, the cHL subtype most closely related to MLBCL. Using quantitative immunohistochemical methods, we document the association between 9p24.1 copy number and PD-1 ligand expression in primary tumors. In cHL and MLBCL, the extended 9p24.1 amplification region also included the Janus kinase 2 (JAK2) locus. Of note, JAK2 amplification increased protein expression and activity, specifically induced PD-1 ligand transcription and enhanced sensitivity to JAK2 inhibition. Therefore, 9p24.1 amplification is a disease-specific structural alteration that increases both the gene dosage of PD-1 ligands and their induction by JAK2, defining the PD-1 pathway and JAK2 as complementary rational therapeutic targets.

Purpose: Galectin-1 (Gal1) is an immunomodulatory glycan-binding protein regulated by an AP1-dependent enhancer in Hodgkin Reed-Sternberg cells. We recently found that Reed-Sternberg cell Gal1 promotes the immunosuppressive T-helper 2/T-regulatory cell–skewed microenvironment in classical Hodgkin lymphoma (cHL). We sought to investigate whether the coordinate expression of activated AP1 pathway components and Gal1 serves as a diagnostic signature of cHL. In addition, because there are common signaling and survival pathways in cHL and additional non–Hodgkin lymphomas, we also evaluated whether the AP1/Gal1 signature is shared by other molecularly or morphologically related lymphomas. Experimental Design: We evaluated 225 cases of primary cHL and non–Hodgkin lymphoma for evidence of a functional AP1/Gal1 signature by immunohistochemical techniques. Results: Gal1 is selectively expressed by malignant Reed-Sternberg cells in >90% of primary cHLs, and Gal1 expression is concordant with the activated AP1 component, c-Jun. In contrast, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, and another Hodgkin-related entity, nodular lymphocyte–predominant Hodgkin lymphoma, do not express Gal1. However, anaplastic large cell lymphoma (ALCL), consistently expresses both Gal1 and its transcriptional regulator, c-Jun. The presence of activated c-Jun, indicative of functional AP1 activity, was confirmed by phospho-c-Jun immunostaining in cHL and ALCL. Conclusions: These findings establish a functional AP1 signature that includes Gal1 expression in cHL and ALCL and suggests a common mechanism for tumor immunotolerance in these diseases. In addition, the combination of Gal1 and c-Jun serve as diagnostic biomarkers that delineate cHL and ALCL from other lymphomas with shared morphologic and/or molecular features.

SummaryB cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

SummaryDiffuse large B cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy, and suggest targeted treatment approaches.

Abstract 1534 + equal contributions Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate and infrequent somatic mutations of TP53 and genes encoding cell cycle pathway components. Despite recent advances in the molecular understanding of DLBCL pathogenesis, clinical models such as the International Prognostic Index (IPI) are still used to identify high-risk patients. By integrating high-resolution high-density SNP array data with transcriptional profiles and performing pathway analysis in 180 primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary DLBCLs with single copy loss of 17p13.1 (TP53/RPL26/KDM6B) often had CNAs of an additional p53 modifier – 9p21.3 (CDKN2A/ARF), 19q13.42 (BCL2L12), 12q15 (MDM2) or 1q23.3 (MDM4/RFWD2). CNAs of the respective p53 modifiers, CDKN2A (ARF, 9p21.3), MDM2 (12q15) and MDM4/RFWD2 (1q23.3), occurred in largely separate groups of tumors. DLBCLs with CNAs of p53 pathway members frequently exhibited concurrent alterations of additional cell cycle components such as CCND3 (6p21.32), CDK6 (7q22.1), CDK2/CDK4 (12q15) and/or RB1 (13q14.2) or RBL2 (16q12.2). When the primary DLBCLs were clustered in the space of CNAs that perturb p53 pathway and cell cycle components, 66% of tumors had multiple alterations (“complex” pattern) whereas the remaining 34% of tumors lacked these lesions (“clean” signature). Tumors with “complex” alterations of p53 pathway and cell cycle components also had more total CNAs and more frequent TP53 mutations, highlighting the association between p53 deficiency, cell cycle deregulation and increased genomic instability in DLBCL. The patterns of “complex” vs. “clean” CNAs of p53 pathway and cell cycle components and the association between the “complex” signature and total CNAs were confirmed in an independent series of primary DLBCLs. To further characterize DLBCLs with “complex” vs. “clean” CNA patterns, we performed gene set enrichment analyses with publicly available series of p53 target genes and a RB-deficiency gene set which included multiple E2F targets. The p53 target transcripts were significantly less abundant in “complex” DLBCLs, directly linking their genetic signature of p53 deficiency with decreased p53 activity. Furthermore, the RB-deficiency gene set was significantly enriched in “complex” DLBCLs suggesting that these tumors had increased E2F-mediated cell cycle progression. Consistent with these observations, DLBCLs with “complex” CNA patterns also had significantly higher proliferation indices as determined by Ki67 immunostaining. We next assessed the prognostic significance of the “complex” CNA pattern in the subset of patients who were treated with R-CHOP (rituxan, cyclophosphamide, adriamycin, oncovin, prednisone) and had long-term follow-up. R-CHOP treated patients with “complex” CNA patterns had a 5-year overall survival of only 62%; in contrast, those with “clean” CNA signatures had an 100% overall survival (p =.001). The association between CN complexity and outcome was independent of transcriptionally defined categories and additive to the clinical IPI risk model. Taken together, these data provide a structural basis for deregulated cell cycle, increased cellular proliferation and unfavorable outcome in DLBCL and suggest targeted treatment strategies. Disclosures: No relevant conflicts of interest to declare.

Abstract 746 Classical Hodgkin Lymphoma (cHL) Reed-Sternberg (RS) cells have crippling mutations of their rearranged Ig genes and lack B-cell receptor-mediated survival signals. As a consequence, cHLs rely on alternative survival and proliferation pathways including AP-1. Although cHL RS cells express high levels of the AP-1 components, cJun and JunB, and depend on AP-1-mediated proliferation signals, the mechanism of constitutive AP-1 activation in cHL remains undefined. In a screen for genetic abnormalities in cHL, we integrated microRNA (miR) expression profiles with high resolution copy number data and identified single copy loss of the miR-15a/16-1 locus (chromosome 13q14) and decreased miR-15a/16 expression in 75% of examined HL cell lines. We isolated primary Hodgkin RS cells using laser-capture microdissection and confirmed frequent single copy loss of the miR-15a/16-1 locus by quantitative PCR and associated decreased miR-15a/16 abundance by RT-PCR in 10/23 (43%) of primary tumors. Using retroviral vectors containing miR-15a and miR-16, we reconstituted miR-15a/16 expression in HL cell lines with single copy loss of chromosome 13q14. In these HL lines, miR-15a/16 reconstitution was associated with cell cycle arrest, significantly decreased cellular proliferation and induction of apoptosis (88% apoptotic cells at 48 hours). In addition, miR-15a/16 reconstitution resulted in significant downregulation of multiple survival pathway components and known miR-15a/16 targets including panAKT, BCL2 and cyclin D1. Using a stand-alone miRanda algorithm with direct sequence entry, we also identified a candidate miR-15a/16 binding site in the AP-1 component, cJun. In multiple HL cell lines, miR-15a/16 reconstitution decreased the expression of cJun and additional known AP-1 targets such as galectin-1. Therefore, miR-15a/16-1 deletion is a frequent genetic abnormality in HL cell lines and primary tumors, a candidate mechanism of AP-1 activation in HL and a modulator of multiple critical survival pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

Multiple studies have shown the importance of PI3K/Akt/mTOR pathway in the pathogenesis of multiple myeloma (MM). Specifically, it mediates cell proliferation and development of drug resistance. Identifying mTOR as a key kinase acting downstream of Akt led to the prediction that rapamycin, a universal inhibitor of mTORC1-dependent S6K1 phosphorylation, may be useful in the treatment of MM. Cumulative evidence support the hypothesis that rapamycin-induced cytotoxicity is predominantly triggered as a consequence of autophagy (programmed cell death type II) via excessive cell digestion. Therefore activated Akt can be a key upstream inhibitor of two cell death-inducing events: autophagy via mTOR activation and apoptosis via phosphorylation of BAD and inhibition of the catalytic subunit of caspase-9. Importantly, recent studies have demonstrated the existence of strong positive feedback loops from mTOR/S6K1 to Akt, resulting in Akt activation in some cancer types. We confirmed that suppression of mTOR signaling by rapamycin was associated with upregulation of Akt phosphorylation, which may reduce sensitivity to rapamycin. We therefore hypothesized that inhibition of this positive feedback by a potent Akt inhibitor perifosine could augment rapamycin-induced cytotoxicity in MM cells. As expected, perifosine inhibited rapamycin-induced p-Akt, associated with synergistic cytotoxicity, even in the presence of IL-6, IGF-1 or bone marrow stromal cells. Synergism was confirmed by the Chou-Talalay model, demonstrating that a combination index was < 1 over a range of doses evaluated. We further examined the molecular mechanisms whereby this combination treatment induced synergistic cytotoxicity. We first studied its effect on ultrastructure in MM cells assessed by electron microscopy, demonstrating that rapamycin-induced autophagy was augmented by perifosine in MM.1S cells. We also confirmed autophagy by immunofluorescent staining of LC3, an autophagosome membrane-associated protein. Interestingly, both autophagy and apoptosis were observed when cells were cultured for longer periods with rapamycin and perifosine co-treatment. The apoptotic effect of the combination was further assessed by Annexin/PI flow cytometric analysis and caspase/PARP cleavage. Neither inhibition of autophagy by 3-MA, nor the blockade of apoptosis by Z-VAD-FMK rescued MM cell death from the combination treatment. Finally, we demonstrate that the combination of nanoparticle albumin-bound (nab) -rapamycin and perifosine had significant antitumor activity in an in vivo human MM cell xenograft murine model, associated with prolonged survival. We observed pAkt upregulation induced by rapamycin, which was inhibited by perifosine evidenced by immunohistochemical analyses on tumor tissue from the xenograft model. Importantly, autophagy and apoptosis were also confirmed by LC3 and caspase 3 staining on tumor tissue. Collectively our data suggest that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine co-treatment induces autophagy and apoptosis, resulting in synergistic cytotoxicity in MM, providing the rationale for use of this combination in future clinical trial in patients with MM.