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Background: We have previously demonstrated activity of the new Akt inhibitor perifosine (NSC 639966; Keryx, NY) in Waldenstrom’s Macroglobulinemia (WM). Perifosine induced complete inhibition of Akt phosphorylation along with induction of apoptosis in WM cells. However, MAPK pathways and PKC proteins were activated in response to perifosine. MAPK and PKC pathways are known to promote cell proliferation. Therefore, understanding the mechanism by which perifosine induces MEK/ERK and PKC activation is important to better understand the mechanisms of response/resistance to this novel agent in ongoing clinical trials. Methods: WM cell lines (BCWM.1, WM-WSU) were treated with perifosine or with the specific Akt inhibitor Triciribine (Biomol, PA). In addition, knockdown of Akt was performed using shRNA silencing techniques (lentivirus shRNA vector, Invitrogen, Ca). The following inhibitors were also used: PI3K inhibitor LY294002 (Calbiochem, CA) and MEK inhibitor (U0126, Calbiochem, CA). Inhibition of proliferation was measured using the MTT assay. Immunoblotting was performed at different time points. Results: Perifosine induced cytotoxicity in WM cells and induced MEK/ERK activation and pPKC activation in a dose and time dependent fashion. We then treated WM cells with perifosine in the presence or absence of the MEK inhibitor U0126 and demonstrated that the combination of the two agents induced significant synergistic activity. We sought to identify the molecular mechanism by which perifosine induces MEK/ERK activation. We demonstrated that the specific AKT inhibitor Triciribine inhibited AKT and induced cytotoxicity in WM cells in a similar fashion to perifosine. However, unlike perifosine, it did not enhance MEK/ERK activity. Similarly, using Akt shRNA, we demonstrated that, despite inhibition of Akt activation, MEK/ERK was not activated. These data indicate that the effect of perifosine on MEK/ERK pathway is not through a compensatory feedback mechanism of Akt inhibition as previously thought. Therefore, we hypothesized that the effect of perifosine on the MEK/ERK pathway is through modulation of upstream pathways, specifically PI3K, PKC and c-Raf/MEK pathways. We first demonstrated that the specific PI3K inhibitor LY294002 (25mM for 15 minutes) completely abrogated Akt phosphorylation, while inducing significant ERK activation, indicating that the effect of perifosine on MEK/ERK may be similar to that of LY294002. We also demonstrated that perifosine and LY294002 activated c-Raf and pan-pPKC at 4 hrs. Conclusion: Based on this, we believe that in the presence of perifosine, growth receptor stimulation leads to PLC and RTK activation, which induces PIP2 stimulation. PIP2 is upstream of PI3K and PKC. Given that PI3K is blocked by perifosine, PIP2 leads to activation of PKC, which then induces growth stimulation, and activation of c-Raf and downstream MEK/ERK. In addition, growth receptors may also activate Raf through the Ras/Raf/MEK pathway, independent of PKC. These studies provide a better understanding of molecular mechanisms that regulate resistance to perifosine. Future combinations of perifosine with MEK inhibitors or PKC inhibitors such as AZD6244 and Enzastaurin may overcome this resistance and induce significant activity in WM.

Background: Waldenstrom’s Macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma with limited options of therapy. The PI3k/Akt pathway is a critical regulator of cell survival. Our previous studies using proteomic analysis have demonstrated upregulation of members of the PI3k/Akt pathway in WM. We examined whether the new Akt inhibitor perifosine (NSC 639966; Keryx, NY) induces cytotoxicity in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Primary CD19+ malignant cells were obtained from patients after informed consent. Inhibition of proliferation was measured using the MTT assay; DNA synthesis was measured using the thymidine uptake assay and apoptosis using Apo2.7 flow cytometry. Bone marrow stromal cells (BMSC) confer growth and resistance to conventional treatments. We therefore, tested the effect of perifosine on WM cells co-cultured with BMSC. Immunoblotting for signaling pathways was performed at different time (30 minutes to 16 hrs) and doses of therapy. In vivo activity of perifosine was assessed using a SCID-irradiated model with subcutaneous tumors in which perifosine was administered by oral gavage daily (35 mg/kg/day). A two-sided t-test was used to determine statistical differences. Results: Perifosine inhibited phosphorylation of Akt in a dose- and time- dependent fashion, as well as downstream GSK3a/b and ribosomal phospho-S6. Perifosine inhibited Akt activity as confirmed by Akt kinase assay. Perifosine induced significant cytotoxicity and inhibition of DNA synthesis with an IC50 of 5-20uM in all cell lines tested. Similar effects were observed in primary CD19+ patient WM cells. Perifosine induced apoptosis in WM cells as demonstrated by flow cytometry. The mechanism of apoptosis induced by perifosine was through activation of SAPK/JNK pathway, followed by caspase-8, -9 and PARP cleavage. The JNK inhibitor SP600125 abrogated perifosine-induced apoptosis. The growth inhibitory effects of perifosine were significant even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, perifosine did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells or in hematopietic stem cells in a methylcellulose colony forming cell assay, indicating lack of toxicity on normal cells. Interestingly, MAPK members such as MEK/ERK were activated by perifosine. The MEK inhibitor U0126 significantly enhanced perifosine-induced cytotoxicity in WM cells, indicating that this combination may be synergistic in vivo. Finally, perifosine induced significant reduction in WM tumor growth in vivo, as compared to control cohort treated with vehicle only at week 6 (p=0.05). Conclusion: Perifosine has significant antitumor activity in WM both in vitro and in vivo. These results provide the framework for clinical evaluation of perifosine in WM. Supported in part by the Leukemia and Lymphoma Society, the Lymphoma Research Foundation and an American Society of Hematology Scholar Award. * XL and XJ are co-first authors.

13134 Background: Perifosine is a novel alkylphospholipid that has been shown to affect multiple signal transduction pathways including Akt, MAPK and JNK (Kondapaka, Mol. Canc. Ther 2: 1093–1103. 2003). Treatment with a taxane initially activates Akt, and persistent activation increases resistance to the drug (Vanderweele, Mol. Canc. Ther. 3: 1605–13, 2004). Methods: Twelve patients (pts) were enrolled on this study. T was given at a dose of 175 mg/m2 on day 8 (after 7 days of perifosine) of a 21 day cycle; this was to obtain a steady state level of P at the tumor before exposure to T. The intent of the protocol was to determine if full dose P could be delivered with 50 mg of perifosine given orally 1, 2 or 3 times a day on days 1–14 of each cycle. Results: Disease sites included lung 3, thyroid 3, breast 1, esophagus 1 and other 4, Median age was 66 (range 45 - 83); 6 pts were male and median ECOG performance status was 1 (range 0–2). All pts had received prior chemotherapy (median 2 regimens); 3 had prior treatment with a taxane. Three pts were entered at each dose level and the cohort expanded to 6 pts if 2 or more pts experienced a grade 3/4 non-hematologic toxicity (DLT) during cycle 1. A dose level was toxic if 4 or more pts experienced a DLT during cycle 1. A total of 30 cycles and a median of 2 cycles (range 1–11) per patient were delivered. There were no grade 3/4 hematologic toxicities. Full dose T was given in all treatment cycles. P dose reductions were required in 6% of cycles (50 mg - 7%, 100 mg - 0%, 150 mg - 7%). One patient missed one dose due to nausea and one pt was stopped due to diarrhea. The grade 3 toxicities for each cohort are given in the table below. The elevated glucose value was 321. Nine pts were evaluable for response; two pts with thyroid cancer had stable disease by the RECIST criteria for 9 and 10+ months. Conclusions: In this study the usual single agent doses of P (150 mg daily) & T (175 mg/m2 q 3 weeks) were given together without increasing the toxicities that would be expected from using each drug alone. Phase II studies are warranted to define activity of the combination. [Table: see text] [Table: see text]

13063 Background: Perifosine (P) is known to modulate signal transduction pathways, including Akt which is frequently constitutively activated in NSCLC. The half life of P is ∼100 hours and GI toxicities are dose limiting. Studies of a single dose weekly regimen found that the maximum tolerated dose (MTD) was 800 mg. This study was undertaken to see if a higher MTD could be reached using divided doses in NSCLC patients. Methods: Twenty NSCLC patients (pts) were given P, 300 mg, at intervals of 4–6 hours with no more than 4 doses in a 24 hour period. The starting dose was 900 mg and this was escalated by 300 mg/week in subsequent pts to an MTD of 1800 mg. Results: The median age of pts was 62 (range 39–79); 9 were male and median ECOG performance status was 1 (range 0–1). Nineteen pts had received prior chemotherapy (median 2 regimens) and 18 prior radiotherapy. Three pts were entered at each dose and the cohort expanded to 6 pts if 1 or more experienced a grade 3/4 non-hematologic toxicity (DLT) during the first week of therapy. A dose level was toxic if 2 or more pts experienced a DLT. The median time on study was 8 weeks (range 2–28). Seventy-seven percent of pts had no dose reduction (85% of those on 900 mg, 95% on 1200, 72% on 1500, and 78% on 1800 mg). In cohort 1, one pt died within a week without toxicity from P and 1 pt experienced grade 3 nausea, so this cohort was expanded. The other pts in the cohort had no toxicity. In cohort 4 one pt. took 300 mg daily for 6 days and was considered inevaluable. In cohort 3, grade 3/4 toxicity was not observed until after day 28 in 3 pts. Fifteen pts were evaluable for response; 1 had an unconfirmed partial response and 2 had stable disease. Conclusions: This study demonstrated that higher doses of P can be administered on a weekly schedule with divided doses. Weekly doses of 900 and 1200 mg lead to almost no toxicity in most pts. This study has been expanded to a phase 2 trial with direct comparison of weekly and daily doses. The grade 3/4 toxicities for each cohort are given in the table below. [Table: see text] [Table: see text]

13084 Background: Miltefosine (M) has been shown to be synergistic with gemcitabine (G) in vitro (Georgieva Cancer Letters 182: 163–174, 2002). Perifosine (P) is a derivative of M with less GI toxicity. Methods: Twenty-two patients (pts) were enrolled on a phase 1 study to determine if G could be given at a dose of 1000 mg/m2 on days 1 and 8 of a 21-day cycle with 50 mg of P given orally 1, 2 or 3 times a day on days 1–14. Three pts were entered at each dose and the cohort expanded to 6 if 2 or more experienced a grade 3/4 non-hematologic toxicity (DLT) during the first cycle. A dose was toxic if 4 or more pts experienced a DLT during cycle 1. Ten additional pts were entered at the highest dose achieved. Results: Disease sites: lung 6, pancreas 3, breast 3, renal 2, sarcoma 2, other 5. The median age was 61 (range 29–82); 11 pts were male and median ECOG performance status was 1 (range 0–2). All pts had received prior chemotherapy (median 3, range 1–4) & 6 had received prior G. 10 pts had received prior radiotherapy. The grade 3/4 non hematologic toxicities for each cohort are given in the table below: One pt on cohort 3 died from pneumonia and sepsis unrelated to therapy after cycle 1. A median of 4 cycles (range 1 - 18) were delivered. At each dose 50% of pts had no G dose reductions. P doses were reduced in 7% of cycles (50 mg - 5%, 100 mg - 15%, 150 mg - 7%). Fifteen pts were evaluable & 4 partial responses (lung, breast, pancreas and unknown primary) were seen; 6 pts had stable disease (renal (2), sarcoma (2), liver and head/neck) for a median of 6 (range 3 - 9) months. No responder had received prior G. Pharmacokinetic (PK) studies showed that the steady state level at 150 mg was 9.47 ± 4.56 μM compared to 10.14 ± 2.02 μM in an earlier study (Crul, Eur. J. Cancer 38: 1615–21, 2002) when this dose of perifosine was used alone. Conclusions: It is concluded that full dose G can be safely administered with perifosine doses up to 150 mg with manageable toxicity and without affecting the PK of P. Phase II studies are warranted to further define the activity of the combination and the contribution of P. [Table: see text] [Table: see text]

13142 Background: Miltefosine (M) is an alkophosholipid with activity as a topical treatment for cutaneous breast cancer lesions. Severe GI toxicity limited its development as a systemic anticancer treatment. P is an equipotent derivative of M with less GI toxicity, but nausea,(N), vomiting (V) & diarrhea (D) are still dose limiting. Methods: This phase 1 study evaluated whether the addition of Pr would enhance the tolerability of P & T in combination. Twenty-five patients (pts) were enrolled. The dose of T was 75 mg/m2 on day 8 of a 21 day cycle and half of the pts were randomized to also receive Pr, 5 mg bid. 50 mg of P was given orally 1, 2 or 3 times a day in successive cohorts on days 1 - 14 of each cycle. Results: Disease sites included lung 9, prostate 7, pancreas 3, ovary 2, breast 1 and other 4. The median age was 63 (range 41 - 80). 18 pts were male & median ECOG performance status was 1 (range 0–2). 22 pts had received a median of 2 prior chemotherapy regimens, 15 a prior taxane and 16 prior radiotherapy. Forty-two cycles of T+P were given without Pr (median 3 & range 1 - 18 per pt) and 84 cycles were given with Pr (median 4.5 cycles & range 1 - 17 per pt). The percentage of pts experiencing various grades (Gr) of N, V & D without and with prednisone are shown in the table below: Five of 12 pts not receiving Pr had gr 2 or 3 toxicities compared to 1 of 13 given Pr. (p [Table: see text] [Table: see text]

Perifosine is an alkylphosphocholine with structural similarity to phospholipids that are the main constituents of cellular membranes. It is the first orally available compound inducing AKT-inhibition in clinical trials. In myeloma tumor cells, Perifosine can inhibit Akt and induce apoptosis. Perifosine inhibits the growth of myeloma tumor cells in vivo and prolongs survival in a murine myeloma model. Triple immune deficient female beige-nude-xid (BNX) mice were inoculated with subcutaneous myeloma tumors derived from MM.1S cells. When measurable growing tumors appeared, mice were randomized to receive Perifosine via oral gavage at either 250mg/kg/week in two divided doses or 36mg/kg/day, or vehicle control (PBS). Animals were sacrificed when tumors reached 2cm or if they appeared moribund. Tumor growth was evaluated using caliper measurements from the first day of treatment until day of first sacrifice, which was day 15 for controls, day 32 for daily treated and day 30 for weekly treated. The tumors of control mice all reached 2cm by day 60, whereas 71% of daily treated mice and 57% of weekly treated mice remained alive at this time, a significantly improved survival amongst treated mice (p=0.01). Surviving treated mice were sacrificed on day 81 for further histological and molecular analysis. Terminal blood was obtained from 4 control mice, 4 daily-treated mice, and 4 weekly-treated mice at the time of sacrifice. Peripheral blood was analyzed with an automated Coulter counter and differential counts were confirmed with a blood smear. In control mice, the mean hemoglobin was 12g/dl, white cell count 7x109/l, and mean platelet count was 292x109/l. In contrast, the respective values for weekly-treated mice were 11, 9 and 944; and for daily treated mice were 10, 11 and 752. The increase in white cell count was due predominantly to a neutrophilia, except in one weekly treated mouse a monocytosis was observed. Blood smears of treated mice confirmed neutrophilia, including a left shift and band donut cells, with thrombocytosis. In placebo-treated animals, splenic architecture consisted of a normal distribution of red pulp and white pulp. The marrow consisted of a normal distribution of hematopoietic cells, with red cell precursors, lymphocytes, maturing myeloid cells and megakaryocytes. There was a normal amount of fat throughout the marrow. In contrast, the splenic architecture of treated mice was partially effaced by a marked expansion of white pulp comprised of maturing myeloid cells and scattered admixed megakaryocytes. The marrow of treated mice displayed marked hypercellularity, consisting predominantly of myeloid hyperplasia, donut cells and band neutrophils. Histological examination of tumors revealed large areas with distinct histological features, characterized by large abnormal foamy cells not seen in control mice, giving a “popcorn lacunae” characteristic. Neutrophils could be observed within tumor blood vessels and band forms and donut cells were found infiltrating the tumors. These findings have clinical relevance because myeloid suppression is a dose-limiting toxicity of many cytotoxic agents, and myeloid hyperplasia is usually only observed in the setting of growth factor stimulation. Coupled with its remarkable in vitro myeloma cytotoxicity, these results strongly support the use of Perifosine in clinical trials for patients with multiple myeloma.

Prior studies have demonstrated that MM cells with PTEN mutation and high AKT activity are more sensitive to inhibitors of the PI3K/Akt/mTOR pathway. However, the molecular mechanisms that regulate the differential response to these agents are not well characterized. The objective of this study was to determine proteins that are different between MM cell lines with higher AKT activity (OPM2) and lower AKT activity (MM.1S) in response to the AKT inhibitor, perifosine. Methods: MM cell lines (MM.1S and OPM2) were treated with serial concentration of perifosine (KRX-0401, Keryx, NY, NY, provided by the NCI). Proteomic analysis using the nanoscale BD Clontech antibody-based protein microarray technique was performed using cells treated with perifosine (10uM for 16 hrs) or vehicle (sterile water) as control. Apoptosis was determined using Annexin V/PI FACS analysis at 24 and 48 hrs. The treatment time and concentration were chosen so that it did not induce more than 25% apoptosis to ensure adequate analysis of changes in signaling pathways. The antibody microarray is a technique that detects differences in protein abundance between the treated and control sample with each experiment by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 human monoclonal antibodies. Two microarray slides were used for each experiment. The slides were scanned using the Axon GenePix 4000B scanner. Two ratios were generated from the spot images for each protein target. The mean of the ratios of Cy5/Cy3 of both slides were analyzed using Clontech software and used to calculate an Internally Normalized Ratio (INR = (Ratio1/Ratio2, ratios 1 and 2 correspond to slides 1 and 2) for each spot on the array. The INR values were input into GeneSpring 6.0 software (Silicon Genetics, CA). The data was normalized to the mean INR of the two cell lines. Proteins whose expression level changed relative to control greater than 1.3 fold were determined. Unsupervised clustering demonstrated a different protein signature between MM.1S and OPM2 in response to perifosine. There were 144 proteins differentially expressed by 1.3 fold between MM.1S and OPM2. Proteins that were downregulated in OPM2 as compared to MM.1S included those in the PI3K pathway and cell cycle regulation such as PTEN, p70S6Kinase, the AKT substrate GSK-3, eEF-2 kinase, eIF-4g, Ku-80, cyclin A, E2F-2, CDK2, CDK7, and c-myc; proteins involved in apoptosis such as p21WAF, caspase 4 and 8, FADD, and PARP; kinases such as PKAc, PKA RI, ERK2, and JNK1; and other proteins regulating apoptosis and proliferation including p53, the NF-kB inhibitor IkB, and the heat shock protein HSP70. The nanoscale protein array is a useful and rapid technique that may be used to identify differences between resistant and sensitive cells to novel therapeutic agents. We identified proteins that are differentially expressed between MM cells sensitive and relatively resistant to the AKT inhibitor perifosine. Further analysis of the role of these proteins in the mechanism of resistance/sensitivity to perifosine is being performed. Future use of inhibitors of NF-kB (bortezomib) or heat shock protein inhibitors in conjunction with perifosine may overcome resistance induced by these proteins in MM cells with low AKT activity. Supported in part by an ASH scholar award and an MMRF grant.

Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death. Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy. Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone. Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.

The serine-threonine kinase AKT is a mediator of tumor proliferation, and its inhibition leads to induction of apoptosis in MM. Heat shock protein-90 (HSP90) is a chaperone protein involved in the refolding of proteins destabilized by stress, including AKT. HSP90 inhibitors have demonstrated in vitro and in vivo activity in MM, and preliminary activity in a phase I clinical trial in MM. We hypothesized that the combination of agents that target two dysregulated pathways in MM, and that interact at the level of AKT will lead to a synergistic cytotoxic activity in MM. MM cell lines with high level of AKT activity (OPM2) and lower AKT activity (multiple dexamethasone-sensitive MM.1S, dexamethasone-resistant MM.1R, and plasma cell leukemia cell line OPM1) were exposed to serial dilutions of perifosine 2-50uM (KRX-0401, Keryx, NY, NY, provided by the NCI) and 17-DMAG 10-200nM (supplied by NCI) alone and in combination for 48 hrs. Inhibition of proliferation was measured using the MTT proliferation assay. Apoptosis was determined using Annexin V/PI flow cytometry analysis (BD Biosciences, CA). Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method. A two-sided t-test was used to determine differences in response. Perifosine induced a dose dependent inhibition of proliferation in all cell lines tested with 30uM inducing 49% inhibition as compared to control and 50uM inducing 60% inhibition in MM.1S cells. Perifosine 30uM induced more significant apoptosis in cell lines with high AKT activity (OPM2) with 51% apoptosis as compared to 14.7% in MM.1S cells with lower AKT activity (p=0.001). 17-DMAG demonstrated a dose dependent inhibition of proliferation and induction of apoptosis in all cell lines tested with 17-DMAG 100nM inducing 40% inhibition as compared to control and 200nM inducing 56% inhibition in MM.1S. There was no differential response to 17-DMAG in cell lines tested. The combination of 30uM perifosine and 100nM 17-DMAG resulted in a significant inhibition of proliferation with 76% inhibition as compared to each agent alone (p=0.0001, perifosine alone vs. combination). The combination was synergistic with a combination index of 0.1 according to the Chou-Talalay method. Apoptosis analysis at 48 hrs demonstrated 13.9% apoptosis with perifosine 30uM, 3.1% with 17-DMAG 100nM alone, and 47.9% with the combination of the two agents (p=0.004 combination vs. perifosine). The combination of the AKT inhibitor, perifosine and HSP90 inhibitor, 17-DMAG demonstrated a synergistic anti-proliferative and pro-apoptotic effect on MM cell lines as compared to each agent alone. Cell lines with higher AKT activity were more sensitive to the AKT inhibitor, perifosine. Targeting both the PI3kinase pathway and the heat shock protein response represents an attractive approach to future therapeutic options in relapsed/refractory MM where drug resistance is often a major problem. Furthermore, the differential activity noted among higher AKT activity and lower AKT activity cell lines raises the possibility of tailoring therapy based on AKT expression levels in the future.

Introduction Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines. Method The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT. Results As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone. Conclusion Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma. Figure 1 Figure 1. Figure 2 Figure 2.

Homing of plasma cells to the bone marrow microenvironment supports the cells’ survival and growth. We have previously demonstrated that MM cells migrate in response to the chemokine SDF-1 and that the PI3K/AKT pathway is an important regulator of migration in MM. The AKT inhibitor, perifosine (KRX-0401, Keryx, NY, NY, provided by the NCI) is an alkylphosphocholine, a member of a new class of potent AKT inhibitors that is being tested preclinically as a therapeutic agent in MM. The objective of this study was to determine the effect of this novel inhibitor on the migration and adhesion of MM cells and to determine differences in response between MM cells with high AKT activity (OPM2 cells) and lower AKT activity (MM.1S). MM cell lines were treated for 16 hrs with serial concentrations of perifosine 2–50uM or vehicle (sterile water). Apoptosis was determined using Annexin V/PI flow cytometry analysis (BD Biosciences, CA). Migration was determined using a transwell migration assay (Costar, Corning, NY). Treated cells were washed, placed in serum free medium for two hours, then placed in the upper chambers of the migration assay with 1% FCS medium in the presence of the chemoattractant SDF-1 in the lower chambers (SDF-1 20nM, a concentration previously determined to induce maximum migration in MM cells). After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96 well plated coated with fibronectin in the presence or absence of serial concentrations of SDF-1 (10–30nM). A two-tailed student t-test was used for statistical analysis. Perifosine induced a dose dependent inhibition of migration in both MM.1S and OPM2. In MM.1S, 2uM perifosine inhibited migration to 82% while 5uM inhibited migration to 44% of control. There was no further significant reduction of migration at 20nM of perifosine (40% migration). In OPM2, 2uM perifosine inhibited migration to a greater extent than MM.1S at 59% of control (p=0.03), and 5uM induced 41% migration similar to the effect observed in MM.1S (p=0.7). There was no induction of apoptosis up to 48hrs of treatment with the low concentrations of perifosine (2–5uM) indicating that the effect of low dose perifosine on MM cells is specific to migration and not due to apoptosis of cells. We have previously demonstrated that SDF-1 increases adhesion of MM cells to fibronectin indicating activation of alpha4beta integrin receptor, with 30nM SDF-1 inducing 296% increase in adhesion. Perifosine alone (2–20uM) inhibited adhesion of MM cells (38% with 2uM, and 12% with 5–20uM as compared to control). Perifosine 2–20uM inhibited adhesion even in the presence of 30nM SDF-1 (55% to 12% respectively). There was no difference in the adhesion response of MM.1S and OPM2 to perifosine. We demonstrate that low doses of the AKT inhibitor perifosine inhibit migration and adhesion of MM cells in response to SDF-1. These doses do not induce apoptosis, and therefore are specific to inhibiting migration. Future studies to test the AKT inhibitor perifosine as a modulator of MM homing, migration and adhesion in vivo are warranted. Supported in part by an ASH scholar award and an MMRF grant.

4642 Background: Perifosine is an oral alkylphospholipid that inhibits cancer cell growth via decreased AKT phosphorylation. To evaluate its efficacy and tolerability in early stage prostate cancer...

4166 Background: Perifosine is a synthetic, substituted heterocyclic alkylphospholipid, structurally related to miltefosine, with a unique spectrum of activity across the NCI 60 cell line screen. Phase I testing identified a recommended phase 2 dose (RP2D) of 150 mg/dy on a 21/28 day schedule with anorexia, fatigue, dehydration, nausea/vomiting as the predominant toxicities. Methods: Patients had advanced pancreatic adenocarcinoma and were allowed to have one prior systemic therapy. Perifosine [50mg tablets] was given on a 28 day cycle: Cycle 1 -loading dose 1050 mg over 2 days followed by 150mg daily on days 3–21; Cycle 2 and greater -day 1 dose of 300mg followed by 150mg days 2–21. A 2 stage design was employed with continuation to stage 2 only if 4/15 survive 6 months from study entry. In parallel with stage 1 of the clinical study, Panc-1 and MiaPaCa-2 xenografts were treated with perifosine at the MTD of 200mg/kg on Day 1 followed by 20mg/kg daily by oral gavage. Animals were sacrificed at 0, 24, 48,...

3064 Background: ALPs represent a group of anti-cancer drugs that primarily target the plasma membrane affecting both apoptotic and survival signaling. ALPs exert anti-angiogenic effects, act as ra...

Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp2-iminosugar glycolipid (sp2-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO2-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO2-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO2-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO2-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO2-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO2-ONJ in microglia., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness: SAF2015-65267-R (MINECO/FEDER), SAF2016-76083-R (MINECO-FEDER), CTQ2015-64425-C2-1-R (MINECO-FEDER) and CTQ2014-53144-P and grants from the Spanish ISCIII (CIBERdem) and INFLAMES (ISCIII PIE14/00045, co-funded by ERDF, “Investing in your future”). We also acknowledge the European Union project H2020-MSCA-ITN TREATMENT Grant Agreement number: 721236. L.M. thanks the Universitat Autònoma de Barcelona-Banco Santander Program for financial support.

Perifosine is a promising anticancer alkylphospholipid (ALP) that induces apoptosis in tumor cells. Here we report evidences against a role of endocytosis in perifosine uptake by human KB carcinoma cells. We have generated a KB cell line resistant to perifosine (KB PER(R) clone10), which shows cross-resistance to the ALPs miltefosine and edelfosine, a marked impairment in the uptake of (14)C-perifosine at both 37 degrees C and 4 degrees C, and no signs for active efflux of the drug. KB PER(R) clone10 cells show a similar rate of raft-dependent endocytosis with respect to the parental cells, and silencing of both clathrin and dynamin in the latter causes only minor changes in the rate of perifosine uptake. Perifosine uptake is a temperature- and ATP-dependent, N-ethylmaleimide- and orthovanadate-sensitive process in parental cells. Accumulation of (14)C-perifosine and the fluorescent phospholipid analogue 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylethanolamine (NBD-PE) is inhibited by perifosine in a concentration-dependent manner in parental cells. Moreover, NBD-PE accumulation is slower in PER(R) clone10 cells and correlated with phosphatidylserine exposure in their plasma membrane surface. Together, all these data suggest a role of plasma membrane translocation by a putative phospholipid translocase, rather than endocytosis, as the true mechanism for ALPs uptake in KB carcinoma cells.

Introduction: Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and provide an environment for signal transduction proteins which control cell survival and cell death. Altered raft assembly has been implicated in cancer progression. Alkylphospholipids (APL) inhibit the AKT pathway and induce apoptosis of malignant cells but not of their normal counterparts when used at the same concentration. LAT2 (Linker for activation of T-cells family member 2) is a lipid raft component which is disrupted by APLs in leukemic cells. In the present study we evaluated the effect of perifosine, an APL with anticancer activity in humans, on the LAT2 and AKT pathways using a Mantle Cell Lymphoma (MCL) cell line (Granta-519 cells) as a model. We selected MCL cells to study because AKT is constitutively phosphorylated in this disease. Methods: We determined whether perifosine induced apoptosis of unmodified Granta-519 cells using flow cytometry, we measured caspase-3 activity with a synthetic peptide and detected cleaved caspases by western blotting. Then, we demonstrated the participation of LAT2 in AKT signaling. We used a stable knockdown of LAT2 (lentiviral sh-RNA). After transduction, Granta-519 cells were selected using 0.5 µg/mL puromycin for 5 days. Stable LAT2 knockdown reduced LAT2 levels by 90% (Figure 1A) and showed impaired AKT phosphorylation in the absence of ligands (Figure 1B). Results: Perifosine treatment induced apoptosis of Granta-519 cells and the effective dose 50% (ED-50) was 20 μM. Apoptosis was triggered after 6 hours of incubation and activation of the extrinsic and intrinsic pathways of apoptosis was demonstrated. Perifosine decreased LAT2 abundance after 3 hours of treatment, also decreasing its presence in lipid rafts, and led to a decrease in AKT phosphorylation and downstream components of AKT signaling. Moreover, perifosine impaired AKT phosphorylation in the presence of stimuli such as CD40L (330 ng/mL) in Granta-519 cells after a few minutes of incubation, resulting in an effect similar to that of the PI3K inhibitor Wortmannin (1 µM). In vivo studies using Granta-519 subcutaneous xenografts in NSG mice revealed that stable knockdown of LAT2 cells showed reduced tumor growth. sh-RNA CT cells (0.75x106) were injected into the right flank of five mice and the same number of sh-RNA LAT2 cells were injected into the left flank of the same animals. After 3 weeks, the tumor weight (mean + SD) was 0.52 ± 0.37 g for sh-RNA LAT2 tumors compared to 1.41 ± 0.19 g for sh-RNA CT tumors (p=0.0065) (Figure 1C and 1D). The LAT2 content of sh-RNA LAT2 cells and AKT phosphorylation were also reduced (Figure 1E and 1F). Conclusions: These results demonstrate that LAT2 is an important protein for supporting constitutive AKT phosphorylation/activation and downstream signaling in MCL and suggest that inhibition of LAT2 by APLs may have therapeutic applications. Acknowledgments: This research was supported by FAPESP, FINEP, and CNPq. C.H.T. received fellowships from FAPESP Proc. No. 13/07675-3. G.A.F. received a fellowship from CNPq Proc. 150126/2014-0. G.A.F. and C.H.T. contributed equally to this work Figure 1 Figure 1. In vivo evaluation of LAT2 stable knockdown cells using the NSG mouse model: (A) LAT2 stable knockdown was obtained from Granta-519 cells via lentiviral transduction of shRNA that targets LAT2. The cell line identified as “B” was used for further studies. (B) AKT activation was suppressed in LAT2 stable knockdown Granta-519 cells. Both cell lines were maintained with or without FBS for 24 hours. (C) Representative picture of the NSG mouse model xenotransplated with Granta-519. Equal amount of cells were injected into the left (sh-RNA LAT2) and right (sh-RNA CT) thigh of the animal. (D) Comparison of tumors weight: sh-RNA CT (1.408 g SD±0.19) and sh-RNA LAT2 (0.523 g SD±0.37). (E) Representative western blotting analysis of tumor cells against LAT2. (F) AKT phosphorylation was evaluated by western blotting (representative of two animals). Disclosures No relevant conflicts of interest to declare.

Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.

// Alice Cani 1, * , Carolina Simioni 1, * , Alberto M. Martelli 2 , Giorgio Zauli 3 , Giovanna Tabellini 4 , Simona Ultimo 1 , James A. McCubrey 5 , Silvano Capitani 1, 6 , Luca M. Neri 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 3 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy 4 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy 5 Department of Microbiology & Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA 6 LTTA Center, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Luca M. Neri, e-mail: luca.neri@unife.it Silvano Capitani, e-mail: silvano.capitani@unife.it Keywords: T-acute lymphoblastic leukemia, Akt, Perifosine, GSK690693, MK-2206 Received: December 22, 2014 Accepted: January 29, 2015 Published: March 02, 2015 ABSTRACT T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder in which chemotherapy resistance and refractory relapses occur, with a poorer prognostic outcome. Constitutively active PI3K/Akt/mTOR pathway is a common feature of T-ALL upregulating cell proliferation, survival and drug resistance. This pathway is currently under clinical trials with small molecules inhibitors (SMI). To verify whether a multi-inhibition treatment against Akt protein could enhance the efficacy of individual drug administration and overcome drug resistance as well as to obtain a decrease in single drug concentration, we tested on T-ALL cell lines the effects of combined treatments with three Akt inhibitors with different mode of action, GSK690693, MK-2206 and Perifosine. In cells with hyperactivated Akt, combined administration of the drugs displayed a significant synergistic and cytotoxic effect and affected PI3K/Akt/mTOR pathway at much lower concentration than single drug use. Highest synergistic effect for full inhibition of Akt was also related to the timing of every drug administration. Furthermore the triple treatment had greater efficacy in inducing cell cycle arrest in G 0 /G 1 phase and both apoptosis and autophagy. Targeting Akt as a key protein of PI3K/Akt/mTOR pathway with multiple drugs might represent a new and promising pharmacological strategy for treatment of T-ALL patients.

Abstract 3705 INTRODUCTION: Identification of predictive biomarkers of response to molecularly targeted therapy represents a challenging issue. Therefore, we conducted a pharmacodynamic study evaluating phosphorylation levels of ERK (pERK) and AKT (pAKT) in circulating lymphocytes from patients with refractory/relapsed Hodgkin lymphoma (HL) enrolled in two consecutive phase II trials evaluating activity and safety of the multikinase inhibitor Sorafenib (Nexavar®, Bayer) used as single agent, or in combination with the AKT inhibitor Perifosine (Æterna Zentaris GmBH, Germany). METHODS: Between July 2008 and May 2011, 25 patients (15 males and 10 females; median age 36 years, range, 18–73 years) with relapsed (n = 3) or refractory (n = 22) HL were analyzed. In one trial, patients received Sorafenib alone (400 mg BID, per os), and in the second trial Perifosine (50 mg BID, per os) for 4 weeks followed by combination therapy of Perifosine plus Sorafenib (400 mg BID, per os) until disease progression or clinically significant toxicity. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. Peripheral blood lymphocytes (PBL) collected prior to therapy initiation and monthly thereafter were analyzed for pERK and pAKT expression by flow cytometry. Unstimulated or phorbol myristate acetate (PMA)-stimulated PBL were fixed with paraformaldehyde, permeabilized with methanol and stained with Alexa Fluor-conjugated Anti-ERK1/2 (pT202/pY204) and anti-Akt (pS473) monoclonal antibodies (MoAbs) or the appropriate isotype controls (all from Becton-Dickinson). PBL from normal donors were also included as internal control. Results were expressed as percentage of cells stained with the relevant MoAb in PMA-stimulated cultures after subtraction of staining detected in unstimulated PBL. The study was approved by the Institutional Ethical Committee. RESULTS: Eight out of 25 patients (32%) achieved objective responses, including complete response (CR, n = 1) and partial response (PR, n = 7), whereas 9/25 patients achieved stable disease (SD), and 8/25 patients experienced PD. Median overall survival (OS), progression-free survival (PFS) and duration of response (DOR) were 16, 5 and 4 months, respectively. Data of pERK and pAKT were available for 23 and 20 patients, respectively. At baseline, the median pERK level was 43% (range, 6 – 82). Baseline pERK levels were significantly higher in responsive patients, i.e., those achieving CR, PR and SD (n = 14) as compared to unresponsive patients (n = 9), i.e., those experiencing PD, (55 ± 18% vs 22 ± 9%, P =.0001). The median baseline value of pERK efficiently discriminated responsive and unresponsive patients. In fact, 12/12 patients with baseline pERK levels ≥43% achieved CR, PR or SD, whereas 9/11 patients (82%) with baseline pERK CONCLUSIONS: Clinical data from phase II studies using Perifosine and/or Sorafenib in patients with relapsed/refractory HL patients suggest a correlation of baseline pERK and pAKT levels with objective responses and time to tumor progression. Whether these parameters will prove to be useful predictive biomarkers of response in HL patients receiving kinase inhibitors will need to be validated in prospective studies. Disclosures: Off Label Use: Sorafenib in Hodgkin Lymphoma Patients.

Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy., Frontiers in Oncology, 10, ISSN:2234-943X

Background Perifosine is a membrane-targeted alkylphospholipid developed to inhibit the PI3K/Akt pathway and has been suggested as a favorable candidate for combined use with radiotherapy. In this study, we investigated the effect of the combined treatment of perifosine and radiation (CTPR) on prostate cancer cells in vitro and on prostate cancer xenografts in vivo. Methods Human prostate cancer cell line, CWR22RV1, was treated with perifosine, radiation, or CTPR. Clonogenic survival assays, sulforhodamine B cytotoxity assays and cell density assays were used to assess the effectiveness of each therapy in vitro. Measurements of apoptosis, cell cycle analysis by flow cytometry and Western blots were used to evaluate mechanisms of action in vitro. Tumor growth delay assays were used to evaluate radiation induced tumor responses in vivo. Results In vitro, CTPR had greater inhibitory effects on prostate cancer cell viability and clonogenic survival than either perifosine or radiation treatment alone. A marked increase in prostate cancer cell apoptosis was noted in CTPR. Phosphorylation of AKT-T308 AKT and S473 were decreased when using perifosine treatment or CTPR. Cleaved caspase 3 was significantly increased in the CTPR group. In vivo, CTPR had greater inhibitory effects on the growth of xenografts when compared with perifosine or radiation treatment alone groups. Conclusions Perifosine enhances prostate cancer radiosensitivity in vitro and in vivo. These data provide strong support for further development of this combination therapy in clinical studies.