Translation is regulated predominantly at the initiation phase by several signal transduction pathways that are often usurped in human cancers, including the PI3K/Akt/mTOR axis. mTOR exerts unique administration over translation by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex responsible for recruiting 40S ribosomes (and associated factors) to mRNA 5′ cap structures. Hence, there is much interest in targeted therapies that block eIF4F activity to assess the consequences on tumor cell growth and chemotherapy response. We report here that hippuristanol (Hipp), a translation initiation inhibitor that selectively inhibits the eIF4F RNA helicase subunit, eIF4A, resensitizes Eμ-Myc lymphomas to DNA damaging agents, including those that overexpress eIF4E—a modifier of rapamycin responsiveness. As Mcl-1 levels are significantly affected by Hipp, combining its use with the Bcl-2 family inhibitor, ABT-737, leads to a potent synergistic response in triggering cell death in mouse and human lymphoma and leukemia cells. Suppression of eIF4AI using RNA interference also synergized with ABT-737 in murine lymphomas, highlighting eIF4AI as a therapeutic target for modulating tumor cell response to chemotherapy.
hippuristanol; eIF4A; Eμ-Myc; lymphoma; ABT-737; Mcl-1
Next-generation sequencing has led to a revolution in the study of hematological malignancies with a substantial number of publications and discoveries in the last few years. Significant discoveries associated with disease diagnosis, risk stratification, clonal evolution and therapeutic intervention have been generated by this powerful technology. As part of the post-genomic era, sequencing analysis will likely become part of routine clinical testing and the challenge will ultimately be successfully transitioning from gene discovery to preventive and therapeutic intervention as part of individualized medicine strategies. In this report, we review recent advances in the understanding of hematological malignancies derived through genome-wide sequence analysis.
hematological malignancy; genome sequencing; RNA sequencing; clonal architecture
In this study, we have identified the growth factors supporting myeloma self-renewal in eight myeloma cell lines. All cell lines able to form self-colonies displayed constitutive P-AKT and P-ERK1,2 but not P-STAT3 and did not express CD45, suggesting the presence of an insulin-like growth factor 1 (IGF1) loop. We showed that a blocking anti-insulin-like growth factor 1 receptor (IGF1R) monoclonal antibody (mAb) inhibited colony formation in correlation with IGF1R expression and decreased P-AKT. Imatinib or a blocking anti-stem cell factor (SCF) mAb also inhibited colony formation of two cell lines expressing C-KIT and SCF, and decreased P-AKT. Moreover, the PI3K/AKT pathway inhibitor wortmannin inhibited colony formation. Blocking interleukin (IL)6R did not inhibit colony formation in good agreement with a lack of constitutive P-STAT3. We showed that primary cells frequently co-expressed IGF1R/IGF1 but not C-KIT/SCF or IL6R/IL6, suggesting that in vivo autonomous growth could be possible via IGF1R. Despite their similar role in clonogenic growth and shared signaling pathway, IGF1R and C-KIT had opposite prognostic values, suggesting that they were surrogate markers. Indeed, we showed that both C-KIT and IGF1R prognostic values were not independent of MMSET expression. This study highlights the autocrine role of IGF1 in myeloma cells and reinforces the interest in targeting IGF1R in IGFR1+ CD45+/− patients, such as MMSET+ patients.
self-renewal; growth factors; multiple myeloma; IGF1R; C-KIT; AKT
Recent studies have reported an increased risk of second primary malignancies (SPM) following multiple myeloma (MM) diagnosis associated with novel anti-myeloma treatments. We evaluated the risk of SPM among 36 491 MM cases reported to the Surveillance, Epidemiology, and End Results program (SEER) between 1973 and 2008. We calculated overall and site-specific standardized incidence ratio (SIR) and 95% confidence intervals (CI) for 2012 SPM cases diagnosed within the 35-year follow-up. There was no significant overall risk of SPM (SIR=0.98; 95% CI=0.94–1.02); however, there were multiple site-specific risk patterns. The risk of breast and prostate cancer was significantly decreased overall and across age, latency and the year of diagnosis strata. There was an ∼50% increased risk of colorectal cancer 5 years after MM diagnosis (Ptrend<0.001). The risk of hematological malignancies was significantly increased, notably for acute myeloid leukemia (AML; SIR=6.51; 95% CI=5.42–7.83). There was a significant decreasing trend for AML over time, particularly for patients ⩾65. However, no significant change in risk was noted after the introduction of autologous stem cell transplant among younger patients (<65 years). On the basis of observed trends for overall SPM as well as AML, no association between the introduction of novel therapies and SPM following MM has emerged in this large population-based study.
multiple myeloma; second primary malignancies; cancer survival and outcome; SEER
Internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) and nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis, but less is known about their utility to predict outcomes at relapse. We retrospectively analysed outcomes of 70 patients with relapsed, intermediate-risk karyotype AML who received a uniform reinduction regimen, with respect to FLT3-ITD and NPM1 mutation status and first complete remission (CR1) duration. CR1 duration, but not molecular status, was significantly correlated with CR2 rate. On univariate analysis, patients with mutated FLT3-ITD (FLT3+) had significantly worse overall survival (OS) compared with those with neither an NPM1 nor FLT3-ITD mutation (NPM1-/FLT3-). On multivariate analysis, shorter CR1 duration was significantly correlated with inferior OS at relapse (P<0.0001), while FLT3 and NPM1 mutation status and age were not significantly correlated with OS. Patients who subsequently underwent allogeneic stem cell transplant (alloSCT) had a superior OS regardless of CR1 duration, but outcomes were better in patients with CR1 duration>12 months. In intermediate-risk karyotype AML patients receiving reinduction, CR1 duration remains the most important predictor of OS at relapse; FLT3-ITD and NPM1 status are not independent predictors of survival.
acute myeloid leukemia; chemotherapy; FLT3 mutation; NPM1 mutation; allogeneic stem cell transplantation; leukemia
Recent evidence shows that lipid raft membrane domains modulate both cell survival and death. Here, we have found that the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is present in the lipid rafts of mantle cell lymphoma (MCL) cells, and this location seems to be critical for full activation and MCL cell survival. The antitumor lipids (ATLs) edelfosine and perifosine target rafts, and we found that ATLs exerted in vitro and in vivo antitumor activity against MCL cells by displacing Akt as well as key regulatory kinases p-PDK1 (phosphatidylinositol-dependent protein kinase 1), PI3K and mTOR (mammalian TOR) from lipid rafts. This raft reorganization led to Akt dephosphorylation, while proapoptotic Fas/CD95 death receptor was recruited into rafts. Raft integrity was critical for Ser473 Akt phosphorylation. ATL-induced apoptosis appeared to correlate with the basal Akt phosphorylation status in MCL cell lines and primary cultures, and could be potentiated by the PI3K inhibitor wortmannin, or inhibited by the Akt activator pervanadate. Classical Akt inhibitors induced apoptosis in MCL cells. Microenvironmental stimuli, such as CD40 ligation or stromal cell contact, did not prevent ATL-induced apoptosis in MCL cell lines and patient-derived cells. These results highlight the role of raft-mediated PI3K/Akt signaling in MCL cell survival and chemotherapy, thus becoming a new target for MCL treatment.
mantle cell lymphoma; lipid rafts; PI3K/Akt signaling; Akt phosphorylation; apoptosis; synthetic antitumor lipids
An increasing body of evidence supports the important role of adhesion to bone marrow microenvironment components for survival and drug resistance of multiple myeloma (MM) cells. Previous studies suggested that stimulation of Toll-like receptors by endogenous ligands released during inflammation and tissue damage may be pro-tumorigenic, but no studies have been performed in relation to modulation of cell adhesion and drug cytotoxicity. Here, we investigated the effect of TLR1/2 activation on adhesion of human myeloma cells to fibronectin, and their sensitivity to the proteasome inhibitor Velcade. It was found that TLR1/2 activation with Pam3CSK4 increased the cytotoxicity of Velcade in L363, OPM-2 and U266 human myeloma cells. This effect was not related to a decreased adhesion of the cells to fibronectin, but TLR1/2 activation stimulated the caspase-3 activity in Velcade-treated myeloma cells, which may be responsible for the enhanced cell death. Inhibitors of NF-κB and MAPK reduced the stimulatory effect. These findings indicate that TLR activation of MM cells could bypass protective effects of cell adhesion and suggest that TLR signaling may also have antitumorigenic potential.
multiple myeloma; Toll-like receptor; Velcade; fibronectin; apoptosis; Pam3CSK4
The Ras/Raf/MEK/extracellular signal regulated kinase (ERK) (Ras/mitogen-activated protein kinases (MAPK)) signal transduction pathway is a crucial mediator of many fundamental biological processes, including cellular proliferation, survival, angiogenesis and migration. Aberrant signalling through the Ras/MAPK cascade is common in a wide array of malignancies, including multiple myeloma (MM), making it an appealing candidate for the development of novel targeted therapies. In this review, we explore our current understanding of the Ras/MAPK pathway and its role in MM. Additionally, we summarise the current status of small molecule inhibitors of MEK under clinical evaluation, and discuss future approaches required to optimise their use.
multiple myeloma; MEK inhibitors; Ras/MAPK; MEK
HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC50 near therapeutic drug blood levels (8–14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations <40 μℳ. Ritonavir, saquinavir and lopinavir inhibited p-AKT comparable to nelfinavir, and showed similar synergistic cytotoxicity with bortezomib against bortezomib-sensitive cells. Nelfinavir had superior synergistic activity with bortezomib/carfilzomib in particular against bortezomib/carfilzomib-resistant myeloma cells. It inhibited not only the proteasomal β1/β5 active sites, similar to bortezomib/carfilzomib, but in addition the β2 proteasome activity not targeted by bortezomib/carfilzomib. Additional inhibition of β2 proteasome activity is known to sensitize cells for bortezomib and carfilzomib. Nelfinavir has unique proteasome inhibiting activity in particular on the bortezomib/carfilzomib-insensitive tryptic (β2) proteasome activity in intact myeloma cells, and is active against bortezomib/carfilzomib-resistant myeloma cells in vitro.
proteasome; drug resistance; myeloma
Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.
targeted cancer therapy; serine protease; treatment resistance; Serpin B9; immunotoxin
Chronic myelogenous leukemia (CML) evolves from a chronic phase characterized by the Philadelphia chromosome as the sole genetic abnormality and the accumulation of mature cells in peripheral blood into blast crisis, which is characterized by the rapid expansion of myeloid- or lymphoid-differentiation-arrested blast cells. Although ample studies have been conducted on the disease progress mechanisms, the underlying molecular mechanisms of the malignant phenotype transition are still unclear. In this study, we have shown that myofibrillogenesis regulator-1 (MR-1) was overexpressed in blast crisis patients and leukemic cells, but there was little trace expressed in healthy individuals and in most patients in CML chronic phase. MR-1 could inhibit the differentiation of myeloid cells into megakaryocytic lineages and accelerate cell proliferation. The molecular mechanism responsible for these effects was the interaction of MR-1 with MEK, which blocked the MEK/ERK signaling pathway by dephosphorylating MEK. Our results provide compelling and important evidence that MR-1 might act as a diagnostic marker and potential target of CML progression from chronic phase to blast crisis.
MR-1; chronic myeloid leukemia; blast crisis; megakaryocytic differentiation; ERK; MEK
Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient.
multiple myeloma; somatic mutations; whole-exome sequencing; adhesion; receptor tyrosine kinases
Epigenetic silencing of the tumor suppressor gene p15Ink4b (CDKN2B) is a frequent event in blood disorders like acute myeloid leukemia and myelodysplastic syndromes. The molecular function of p15Ink4b in hematopoietic differentiation still remains to be elucidated. Our previous study demonstrated that loss of p15Ink4b in mice results in skewing of the differentiation pattern of the common myeloid progenitor towards the myeloid lineage. Here, we investigated a function of p15Ink4b tumor suppressor gene in driving erythroid lineage commitment in hematopoietic progenitors. It was found that p15Ink4b is expressed more highly in committed megakaryocyte–erythroid progenitors than granulocyte–macrophage progenitors. More importantly, mice lacking p15Ink4b have lower numbers of primitive red cell progenitors and a severely impaired response to 5-fluorouracil- and phenylhydrazine-induced hematopoietic stress. Introduction of p15Ink4b into multipotential progenitors produced changes at the molecular level, including activation of mitogen-activated protein kinase\extracellular signal-regulated kinase (MEK/ERK) signaling, increase GATA-1, erythropoietin receptor (EpoR) and decrease Pu1, GATA-2 expression. These changes rendered cells more permissive to erythroid commitment and less permissive to myeloid commitment, as demonstrated by an increase in early burst-forming unit-erythroid formation with concomitant decrease in myeloid colonies. Our results indicate that p15Ink4b functions in hematopoiesis, by maintaining proper lineage commitment of progenitors and assisting in rapid red blood cells replenishment following stress.
p15Ink4b; hematopoiesis; stem cell; cell fate; differentiation; erythropoiesis
Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34+) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34+ progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository.
Pediatric leukemia survival rates have improved dramatically over the past decades. However, current treatment protocols are still largely ineffective in cases of relapsed leukemia and are associated with a significant rate of chronic health conditions. Thus, there is a continued need for new therapeutic options. Here, we show that mer receptor tyrosine kinase (MerTK) was abnormally expressed in approximately one half of pediatric T-cell leukemia patient samples and T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Stimulation of MerTK by the ligand Gas6 led to activation of the prosurvival proteins Erk 1/2 and Stat5, and MerTK-dependent activation of the STAT pathway in leukemia represents a novel finding. Furthermore, inhibition of MerTK expression increased the sensitivity of T-ALL cells to treatment with chemotherapeutic agents and decreased the oncogenic potential of the Jurkat T-ALL cell line in a methylcellulose colony-forming assay. Lastly, inhibition of MerTK expression significantly increased median survival in a xenograft mouse model of leukemia (30.5 days vs 60 days, P<0.0001). These results suggest that inhibition of MerTK is a promising therapeutic strategy for the treatment of leukemia and may allow for dose reduction of currently used chemotherapeutics resulting in decreased rates of therapy-associated toxicities.
MerTK; receptor tyrosine kinase; T-cell leukemia; xenograft mouse model
Bone marrow (BM) cells depend on their niche for growth and survival. However, the genes modulated by niche stimuli have not been discriminated yet. For this purpose, we investigated BM aspirations from patients with various hematological malignancies. Each aspirate was fractionated, and the various samples were fixed at different time points and analyzed by microarray. Identification of niche-modulated genes relied on sustained change in expression following loss of niche regulation. Compared with the reference (‘authentic') samples, which were fixed immediately following aspiration, the BM samples fixed after longer stay out-of-niche acquired numerous changes in gene-expression profile (GEP). The overall genes modulated included a common subset of functionally diverse genes displaying prompt and sustained ‘switch' in expression irrespective of the tumor type. Interestingly, the ‘switch' in GEP was reversible and turned ‘off-and-on' again in culture conditions, resuming cell–cell–matrix contact versus respread into suspension, respectively. Moreover, the resuming of contact prolonged the survival of tumor cells out-of-niche, and the regression of the ‘contactless switch' was followed by induction of a new set of genes, this time mainly encoding extracellular proteins including angiogenic factors and extracellular matrix proteins. Our data set, being unique in authentic expression design, uncovered niche-modulated and niche-modulating genes capable of controlling homing, expansion and angiogenesis.
gene-expression profile; myeloma; leukemia; contactless gene signature; niche-modulated genes; niche-modulating genes
A previous US study reported poorer survival in children with acute lymphoblastic leukemia (ALL) exposed to extremely low-frequency magnetic fields (ELF–MF) above 0.3 μT, but based on small numbers. Data from 3073 cases of childhood ALL were pooled from prospective studies conducted in Canada, Denmark, Germany, Japan, UK and US to determine death or relapse up to 10 years from diagnosis. Adjusting for known prognostic factors, we calculated hazard ratios (HRs) and 95% confidence intervals (CI) for overall survival and event-free survival for ELF–MF exposure categories and by 0.1 μT increases. The HRs by 0.1 μT increases were 1.00 (CI, 0.93–1.07) for event-free survival analysis and 1.04 (CI, 0.97–1.11) for overall survival. ALL cases exposed to >0.3 μT did not have a poorer event-free survival (HR=0.76; CI, 0.44–1.33) or overall survival (HR=0.96; CI, 0.49–1.89). HRs varied little by subtype of ALL. In conclusion, ELF–MF exposure has no impact on the survival probability or risk of relapse in children with ALL.
leukemia; children; survival; electromagnetic fields; adverse effects; pooled analyses
Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias.
ncRNA; snoRNA; multiple myeloma.
To select an appropriate prognostic model in the treatment of mature T- and natural killer (NK) -cell lymphoma (peripheral T-cell lymphoma (PTCL) and NK-/T-cell lymphoma (NKTCL)) is crucial. This study investigated the usefulness of Ann Arbor staging classification International prognostic index (IPI), prognostic index for T-cell lymphoma (PIT) and International peripheral T-cell lymphoma Project score (IPTCLP). Between 2000 and 2009, 176 patients (122 males) with PTCL and NKTCL were diagnosed and treated from a single institute in Taiwan. The correlation between complete response (CR) rate, 3-year overall survival (OS), early mortality rate and four prognostic models was analyzed. Thirty-one patients received hematopoietic stem cell transplantation (HSCT) and were analyzed separately. Three-year OS rate was 34.7%, and anaplastic large-cell lymphoma harbored better outcome than others. IPI score had the lowest Akaike information criterion value (1081.197) and was the best score in predicting OS and early mortality (P=0.009). Ann Arbor stage classification can predict CR rate more precisely (P=0.006). OS was significantly better in patients who received HSCT, even in patients with unfavorable features compared with chemotherapy alone. All prognostic models were useful to evaluate the outcome of patients with PTCL and NKTCL but IPI score did best in predicting OS in PTCL and PIT score in NKTCL. This study also supported the role of HSCT in patients with high-risk or refractory PTCL or NKTCL.
T-cell lymphoma; prognostic score; hematopoietic stem cell transplantation; Asian population
The outcomes for patients with chronic myeloid leukemia have improved dramatically with the development and availability of BCR–ABL1 tyrosine kinase inhibitors (TKIs) over the past decade. TKI therapy has a superior safety profile compared with the previous standard of care, interferon-α, and most adverse events (AEs) observed with front-line and second-line TKI treatment are managed with supportive care. However, some patients are intolerant to TKI therapy and experience AEs that cannot be managed through dose reduction or symptomatic treatment. Careful management of AEs helps patients to remain adherent with treatment and increases their chances for successful outcomes. Proactive vigilance for potential AEs and treatment strategies that reduce symptom burden will help to minimize patient intolerance. This review discusses the most common AEs associated with intolerance to TKI therapy and treatment strategies to help manage patients at risk for or experiencing these events.
chronic myeloid leukemia; dasatinib; imatinib; intolerance; nilotinib; tyrosine kinase inhibitors