Dysregulation of SWI/SNF Chromatin Remodelers in NSCLC: Its Influence on Cancer Therapies including Immunotherapy.

Lung cancer is the leading cause of cancer death worldwide. Molecularly targeted therapeutics and immunotherapy revolutionized the clinical care of NSCLC patients. However, not all NSCLC patients harbor molecular targets (e.g., mutated EGFR), and only a subset benefits from immunotherapy. Moreover, we are lacking reliable biomarkers for immunotherapy, although PD-L1 expression has been mainly used for guiding front-line therapeutic options. Alterations of the SWI/SNF chromatin remodeler occur commonly in patients with NSCLC. This subset of NSCLC tumors tends to be undifferentiated and presents high heterogeneity in histology, and it shows a dismal prognosis because of poor response to the current standard therapies. Catalytic subunits SMARCA4/A2 and DNA binding subunits ARID1A/ARID1B/ARID2 as well as PBRM1 were identified to be the most commonly mutated subunits of SWI/SNF complexes in NSCLC. Mechanistically, alteration of these SWI/SNF subunits contributes to the tumorigenesis of NSCLC through compromising the function of critical tumor suppressor genes, enhancing oncogenic activity as well as impaired DNA repair capacity related to genomic instability. Several vulnerabilities of NSCLCS with altered SWI/SNF subunits were detected and evaluated clinically using EZH2 inhibitors, PROTACs of mutual synthetic lethal paralogs of the SWI/SNF subunits as well as PARP inhibitors. The response of NSCLC tumors with an alteration of SWI/SNF to ICIs might be confounded by the coexistence of mutations in genes capable of influencing patients' response to ICIs. High heterogenicity in the tumor with SWI/SNF deficiency might also be responsible for the seemingly conflicting results of ICI treatment of NSCLC patients with alterations of SWI/SNF. In addition, an alteration of each different SWI/SNF subunit might have a unique impact on the response of NSCLC with deficient SWI/SNF subunits. Prospective studies are required to evaluate how the alterations of the SWI/SNF in the subset of NSCLC patients impact the response to ICI treatment. Finally, it is worthwhile to point out that combining inhibitors of other chromatin modulators with ICIs has been proven to be effective for the treatment of NSCLC with deficient SWI/SNF chromatin remodelers.

Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation.

DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an in vitro kinase assay. Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).

Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

A Novel Therapeutic Induces DEPTOR Degradation in Multiple Myeloma Cells with Resulting Tumor Cytotoxicity.

Prior work indicates DEPTOR expression in multiple myeloma cells could be a therapeutic target. DEPTOR binds to mTOR via its PDZ domain and inhibits mTOR kinase activity. We previously identified a drug, which prevented mTOR-DEPTOR binding (NSC126405) and induced multiple myeloma cytotoxicity. We now report on a related therapeutic, drug 3g, which induces proteasomal degradation of DEPTOR. DEPTOR degradation followed drug 3g binding to its PDZ domain and was not due to caspase activation or enhanced mTOR phosphorylation of DEPTOR. Drug 3g enhanced mTOR activity, and engaged the IRS-1/PI3K/AKT feedback loop with reduced phosphorylation of AKT on T308. Activation of TORC1, in part, mediated multiple myeloma cytotoxicity. Drug 3g was more effective than NSC126405 in preventing binding of recombinant DEPTOR to mTOR, preventing binding of DEPTOR to mTOR inside multiple myeloma cells, in activating mTOR and inducing apoptosis in multiple myeloma cells. In vivo, drug 3g injected daily abrogated DEPTOR expression in xenograft tumors and induced an antitumor effect although modest weight loss was seen. Every-other-day treatment, however, was equally effective without weight loss. Drug 3g also reduced DEPTOR expression in normal tissues. Although no potential toxicity was identified in hematopoietic or hepatic function, moderate cardiac enlargement and glomerular mesangial hypertrophy was seen. DEPTOR protected multiple myeloma cells against bortezomib suggesting anti-DEPTOR drugs could synergize with proteasome inhibitors (PI). Indeed, combinations of drug NSC126405 + bortezomib were synergistic. In contrast, drug 3g was not and was even antagonistic. This antagonism was probably due to prevention of proteasomal DEPTOR degradation.

Structure-activity relationship study of small molecule inhibitors of the DEPTOR-mTOR interaction.

DEPTOR is a 48kDa protein that binds to mTOR and inhibits this kinase within mTORC1 and mTORC2 complexes. Over-expression of DEPTOR specifically occurs in the multiple myeloma (MM) tumor model and DEPTOR knockdown is cytotoxic to MM cells, suggesting it is a potential therapeutic target. Since mTORC1 paralysis protects MM cells against DEPTOR knockdown, it indicates that the protein-protein interaction between DEPTOR and mTOR is key to MM viability vs death. In a previous study, we used a yeast two-hybrid screen of a small inhibitor library to identify a compound that inhibited DEPTOR/mTOR binding in yeast. This therapeutic (compound B) also prevented DEPTOR/mTOR binding in MM cells and was selectively cytotoxic to MM cells. We now present a structure-activity relationship (SAR) study around this compound as a follow-up report of this previous work. This study has led to the discovery of five new leads - namely compounds 3g, 3k, 4d, 4e and 4g - all of which have anti-myeloma cytotoxic properties superior to compound B. Due to their targeting of DEPTOR, these compounds activate mTORC1 and selectively induce MM cell apoptosis and cell cycle arrest.

Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells.

DEPTOR is a 48 kDa protein that binds to mTOR and inhibits this kinase in TORC1 and TORC2 complexes. Overexpression of DEPTOR specifically occurs in a model of multiple myeloma. Its silencing in multiple myeloma cells is sufficient to induce cytotoxicity, suggesting that DEPTOR is a potential therapeutic target. mTORC1 paralysis protects multiple myeloma cells against DEPTOR silencing, implicating mTORC1 in the critical role of DEPTOR in multiple myeloma cell viability. Building on this foundation, we interrogated a small-molecule library for compounds that prevent DEPTOR binding to mTOR in a yeast-two-hybrid assay. One compound was identified that also prevented DEPTOR-mTOR binding in human myeloma cells, with subsequent activation of mTORC1 and mTORC2. In a surface plasmon resonance (SPR) assay, the compound bound to recombinant DEPTOR but not to mTOR. The drug also prevented binding of recombinant DEPTOR to mTOR in the SPR assay. Remarkably, although activating TORC1 and TORC2, the compound induced apoptosis and cell-cycle arrest in multiple myeloma cell lines and prevented outgrowth of human multiple myeloma cells in immunodeficient mice. In vitro cytotoxicity against multiple myeloma cell lines was directly correlated with DEPTOR protein expression and was mediated, in part, by the activation of TORC1 and induction of p21 expression. Additional cytotoxicity was seen against primary multiple myeloma cells, whereas normal hematopoietic colony formation was unaffected. These results further support DEPTOR as a viable therapeutic target in multiple myeloma and suggest an effective strategy of preventing binding of DEPTOR to mTOR. Cancer Res; 76(19); 5822-31. ©2016 AACR.

SGK Kinase Activity in Multiple Myeloma Cells Protects against ER Stress Apoptosis via a SEK-Dependent Mechanism.

UNLABELLED: To assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress-induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib. IMPLICATIONS: Enhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge.

MNK1-induced eIF-4E phosphorylation in myeloma cells: a pathway mediating IL-6-induced expansion and expression of genes involved in metabolic and proteotoxic responses.

Because multiple myeloma (MM) cells are at risk for endoplasmic reticulum (ER) stress, they require a carefully regulated mechanism to promote protein translation of selected transcripts when proliferation is stimulated. MAPK-interacting kinases (MNKs) may provide this mechanism by enhancing cap-dependent translation of a small number of critical transcripts. We, thus, tested whether MNKs played a role in MM responses to the myeloma growth factor interleukin-6 (IL-6). IL-6 activated MNK1 phosphorylation and induced phosphorylation of its substrate, eIF-4E, in MM lines and primary specimens. MNK paralysis, achieved pharmacologically or by shRNA, prevented MM expansion stimulated by IL-6. A phosphodefective eIF-4E mutant also prevented the IL-6 response, supporting the notion that MNK's role was via phosphorylation of eIF-4E. Both pharmacological MNK inhibition and expression of the phosphodefective eIF-4E mutant inhibited MM growth in mice. Although critical for IL-6-induced expansion, eIF-4E phosphorylation had no significant effect on global translation or Ig expression. Deep sequencing of ribosome-protected mRNAs revealed a repertoire of genes involved in metabolic processes and ER stress modulation whose translation was regulated by eIF-4E phosphorylation. These data indicate MM cells exploit the MNK/eIF-4E pathway for selective mRNA translation without enhancing global translation and risking ER stress.

DEPTOR is linked to a TORC1-p21 survival proliferation pathway in multiple myeloma cells.

We investigated the mechanism by which gene silencing of the mTOR inhibitor, DEPTOR, induces cytoreductive effects on multiple myeloma (MM) cells. DEPTOR knockdown resulted in anti-MM effects in several MM cell lines. Using an inducible shRNA to silence DEPTOR, 8226 MM cells underwent TORC1 activation, downregulation of AKT/SGK activity, apoptosis, cell cycle arrest and senescence. These latter cytotoxic effects were prevented by TORC1 paralysis (Raptor knockdown) but not by over-expression of AKT activity. In addition, DEPTOR knockdown-induced MM death was not associated with activation of the unfolded protein response, suggesting that enhanced ER stress did not play a role. In contrast, DEPTOR knockdown in 8226 cells induced p21 expression, independent of p53, and p21 knockdown prevented all of the cytotoxic effects following DEPTOR silencing. DEPTOR silencing resulted in p21 upregulation in additional MM cell lines. Furthermore, DEPTOR silencing in a murine xenograft model resulted in anti-MM effects associated with p21 upregulation. DEPTOR knockdown also resulted in a decreased expression of p21-targeting miRNAs and transfection of miRNA mimics prevented p21 upregulation and apoptosis following DEPTOR silencing. Use of a shRNA-resistant DEPTOR construct ruled out off-target effects of the shRNA. These results indicate that DEPTOR regulates growth and survival of MM cells via a TORC1/p21 pathway and suggest an involvement of p21-targeted miRNAs.

Brief co-incubation of sperm and oocytes for in vitro fertilization techniques.

BACKGROUND: The in vitro fertilization (IVF) technique is commonly used and is the only treatment option for a proportion of infertile couples. To obtain better outcomes of IVF, it is important to enhance embryo quality by optimizing IVF techniques. In IVF procedures, oocytes and sperm are routinely co-incubated overnight, which may expose oocytes and zygotes to suboptimal culture conditions with increased reactive oxygen species (ROS) produced by sperm in this long term culture. As an attempt to avoid possible detrimental effects on the oocytes from long exposure to sperm, the brief co-incubation insemination protocol was developed. However, despite a number of studies in this area, it is unclear whether brief co-incubation improves the IVF outcomes compared with the standard overnight insemination protocol. OBJECTIVES: This Cochrane review aimed to determine whether brief co-incubation of sperm and oocytes improves outcomes compared with the standard overnight insemination protocol for women undergoing IVF. SEARCH METHODS: We searched the Cochrane Menstrual Disorders and Subfertility Group Register (14 June 2012), Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, 1st quarter), MEDLINE (1948 to 14 June 2012), EMBASE (1989 to 14 June 2012), PsycINFO (1806 to 14 June 2012) and CINAHL (1980 to 26 July 2012). In addition, we searched trials registers, reference lists of articles, conference proceedings (American Society for Reproductive Medicine (ASRM), European Society of Human Reproduction and Embryology (ESHRE)) and contacted experts in the field. SELECTION CRITERIA: We included randomized controlled trials (RCTs) comparing brief co-incubation of gametes with the standard overnight insemination protocol. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed studies for inclusion and trial quality, and extracted data. Disagreements were resolved by discussion with a third author. Statistical analysis was performed using RevMan software. MAIN RESULTS: Eight RCTs with 733 women in total that compared brief co-incubation and the standard insemination protocol were included. Live birth was not reported in the included studies. For ongoing pregnancy rate, there were 127 ongoing pregnancies in two trials including 426 women. The low quality evidence showed that brief co-incubation was associated with an increased ongoing pregnancy rate compared to the standard insemination protocol (pooled odds ratio (OR) 2.42, 95% confidence interval (CI) 1.55 to 3.77; P < 0.0001, I(2) = 0%). Measuring clinical pregnancy rate, there were 93 clinical pregnancies in three trials including 372 women. The low quality evidence showed that brief co-incubation was associated with a significantly higher clinical pregnancy rate than the overnight insemination protocol (pooled OR 2.36, 95% CI 1.45 to 3.85; P = 0.0006, I(2) = 0%). For the miscarriage rate, there were six miscarriages in one trial including 167 women. This low quality evidence suggested no significant difference in the odds of miscarriage between brief co-incubation and standard insemination (OR 1.98, 95% CI 0.35 to 11.09; P = 0.44). AUTHORS' CONCLUSIONS: This review has provided evidence that brief co-incubation of sperm and oocytes may improve the ongoing pregnancy and clinical pregnancy rates for infertile women undergoing IVF cycles. More RCTs are required to assess whether brief co-incubation would contribute to a higher live birth rate and a lower miscarriage rate compared to the standard overnight insemination protocol.

Metabolomics identifies pyrimidine starvation as the mechanism of 5-aminoimidazole-4-carboxamide-1-ß-riboside-induced apoptosis in multiple myeloma cells.

To investigate the mechanism by which 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAr) induces apoptosis in multiple myeloma cells, we conducted an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26-fold increase in orotate associated with a decrease in uridine monophosphate (UMP) levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and 5-phosphoribosyl-α-pyrophosphate (PRPP). As all pyrimidine nucleotides can be synthesized from UMP, this suggested that the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine, and thymidine, but not purines adenosine or guanosine, rescued multiple myeloma cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan, or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identified by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back. Although phosphorylation of AICAr by adenosine kinase was required to induce multiple myeloma cell death, apoptosis was not associated with AMP-activated kinase activation or mTORC1 inhibition. A possible explanation for inhibition of UMP synthase activity by AICAr was a depression in cellular levels of PRPP, a substrate of UMP synthase. These data identify pyrimidine biosynthesis as a potential molecular target for future therapeutics in multiple myeloma cells.

Mammalian target of rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression and angiogenesis.

We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

The PP242 mammalian target of rapamycin (mTOR) inhibitor activates extracellular signal-regulated kinase (ERK) in multiple myeloma cells via a target of rapamycin complex 1 (TORC1)/eukaryotic translation initiation factor 4E (eIF-4E)/RAF pathway and activation is a mechanism of resistance.

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

IL-6-induced enhancement of c-Myc translation in multiple myeloma cells: critical role of cytoplasmic localization of the rna-binding protein hnRNP A1.

Prior work indicates that IL-6 can stimulate c-Myc expression in multiple myeloma (MM) cells, which is independent of effects on transcription and due to enhanced translation mediated by an internal ribosome entry site in the 5'-UTR of the c-Myc RNA. The RNA-binding protein hnRNP A1 (A1) was also critical to IL-6-stimulated translation. Because A1 shuttles between nucleus and cytoplasm, we investigated whether the ability of IL-6 to enhance Myc translation was mediated by stimulation of A1 shuttling. In MM cell lines and primary specimens, IL-6 increased A1 cytoplasmic localization. In contrast, there was no effect on the total cellular levels of A1. Use of a dominant negative A1 construct, which prevents endogenous A1 from nucleus-to-cytoplasm transit, prevented the ability of IL-6 to enhance Myc internal ribosome entry site activity, Myc protein expression, and MM cell growth. IL-6-stimulated cytoplasmic localization was mediated by alterations in the C-terminal M9 peptide of A1, and this correlated with the ability of IL-6 to induce serine phosphorylation of this domain. A p38 kinase inhibitor prevented IL-6-induced A1 phosphorylation. Thus, IL-6 activates c-Myc translation in MM cells by inducing A1 phosphorylation and cytoplasmic localization in a p38-dependent fashion. These data suggest A1 as a potential therapeutic target in MM.

Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor.

Although preclinical work with rapalogs suggests potential in treatment of multiple myeloma (MM), they have been less successful clinically. These drugs allostearically inhibit the mammalian target of rapamycin kinase primarily curtailing activity of the target of rapamycin complex (TORC)1. To assess if the mammalian target of rapamycin within the TORC2 complex could be a better target in MM, we tested a new agent, pp242, which prevents activation of TORC2 as well as TORC1. Although comparable to rapamycin against phosphorylation of the TORC1 substrates p70S6kinase and 4E-BP-1, pp242 could also inhibit phosphorylation of AKT on serine 473, a TORC2 substrate, while rapamycin was ineffective. pp242 was also more effective than rapamycin in achieving cytoreduction and apoptosis in MM cells. In addition, pp242 was an effective agent against primary MM cells in vitro and growth of 8226 cells in mice. Knockdown of the TORC2 complex protein, rictor, was deleterious to MM cells further supporting TORC2 as the critical target for pp242. TORC2 activation was frequently identified in primary specimens by immunostaining for AKT phosphorylation on serine 473. Potential mechanisms of up-regulated TORC2 activity in MM were stimulation with interleukin-6 or insulin-like growth factor 1, and phosphatase and tensin homolog or RAS alterations. Combining pp242 with bortezomib led to synergistic anti-MM effects. These results support TORC2 as a therapeutic target in MM.

Effect of autophagy on multiple myeloma cell viability.

Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress-inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib.

Regulation of D-cyclin translation inhibition in myeloma cells treated with mammalian target of rapamycin inhibitors: rationale for combined treatment with extracellular signal-regulated kinase inhibitors and rapamycin.

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal-regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant "low-AKT" myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in "high-AKT" myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity.

IL-6-induced stimulation of c-myc translation in multiple myeloma cells is mediated by myc internal ribosome entry site function and the RNA-binding protein, hnRNP A1.

Prior work indicates that c-myc translation is up-regulated in multiple myeloma cells. To test a role for interleukin (IL)-6 in myc translation, we studied the IL-6-responsive ANBL-6 and IL-6-autocrine U266 cell lines as well as primary patient samples. IL-6 increased c-myc translation, which was resistant to rapamycin, indicating a mechanism independent of mammalian target of rapamycin (mTOR) and cap-dependent translation. In contrast, the cytokine enhanced cap-independent translation via a stimulatory effect on the myc internal ribosome entry site (IRES). As known IRES-trans-activating factors (ITAF) were unaffected by IL-6, we used a yeast-three-hybrid screen to identify novel ITAFs and identified hnRNP A1 (A1) as a mediator of the IL-6 effect. A1 specifically interacted with the myc IRES in filter binding assays as well as EMSAs. Treatment of myeloma cells with IL-6 induced serine phosphorylation of A1 and increased its binding to the myc IRES in vivo in myeloma cells. Primary patient samples also showed binding between A1 and the IRES. RNA interference to knock down hnRNP A1 prevented an IL-6 increase in myc protein expression, myc IRES activity, and cell growth. These data point to hnRNP A1 as a critical regulator of c-myc translation and a potential therapeutic target in multiple myeloma.

Targeting aurora kinases as therapy in multiple myeloma.

The aurora kinases facilitate transit from G2 through cytokinesis and, thus, are targets in cancer therapy. Multiple myeloma (MM) is a malignancy characterized by genetic instability, suggesting a disruption of checkpoints that arrest cells at G2M when injury to the mitotic machinery occurs. Since deficient checkpoints would prevent cell cycle arrest and may render cells susceptible to apoptosis in mitosis and since aurora kinases are intermediaries in checkpoint pathways, we tested antimyeloma effects of 2 agents that inhibit aurora kinases. Both inhibited growth of MM lines and primary myeloma samples at nanomolar concentrations while having less of an effect on proliferating lymphocytes and hematopoietic cells. MM cells were not protected by IL-6 or activating mutations of Ras. Antimyeloma effects included induction of tetraploidy followed by apoptosis. Apoptosis correlated with inhibition of aurora activity as shown by reduction of histone 3B phosphorylation. Ectopic expression of aurora A protected MM cells against aurora inhibitors but had no effect on apoptosis induced by bortezomib. As expression of RHAMM in MM contributes to genetic instability, we tested effects of RHAMM. RHAMM overexpression enhanced sensitivity to apoptosis and RHAMM silencing decreased sensitivity. These results suggest potential for aurora kinase inhibitors in MM especially in patients in whom RHAMM is overexpressed.

Oncogenic RAS mutations in myeloma cells selectively induce cox-2 expression, which participates in enhanced adhesion to fibronectin and chemoresistance.

Oncogenic RAS expression occurs in up to 40% of multiple myeloma (MM) cases and correlates with aggressive disease. Since activated RAS induces cyclooxygenase-2 (cox-2) expression in other tumor models, we tested a role for cox-2 in mutant RAS-containing MM cells. We used the ANBL-6 isogenic MM cell lines in which the IL-6-dependent parental line becomes cytokine independent following transfection with mutated N-RAS or K-RAS. Both mutated N-RAS- and K-RAS-expressing ANBL-6 cells demonstrated a selective up-regulation of cox-2 expression and enhanced secretion of PGE2, a product of cox-2. Furthermore, in 3 primary marrow specimens, which contained MM cells expressing mutated RAS, 15% to 40% of tumor cells were positive for cox-2 expression by immunohistochemistry. We used cox-2 inhibitors, NS398 and celecoxib, and neutralizing anti-PGE2 antibody to test whether cox-2/PGE2 was involved in the aggressive phenotype of MM ANBL-6 cells containing mutated RAS. Although these interventions had no effect on IL-6-independent growth or adhesion to marrow stromal cells, they significantly inhibited the enhanced binding of mutant RAS-containing MM cells to fibronectin and the enhanced resistance to melphalan. These results indicate a selective induction of cox-2 in MM cells containing RAS mutations, which results in heightened binding to extracellular matrix protein and chemotherapeutic drug resistance.