bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of B-CLL Transcriptomic and Proteomic Profiles: Differences between Molecular Subgroups.
Authors: Lewintre Eloisa Jantus , Marin Miguel , Reinoso Cristina , Montaner David , Dopazo Joaquín , Calvete Juan Jose , Garcia-Conde Javier
Published: October 2019

INTRODUCTION: B cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder with a variable clinical course. Patients (pts) with unmutated (unmut) IgVH gene show a shorter progression free survival and overall survival than the patients with IgVH mutated (mut). To understand the differences between molecular subgroups of B-CLL we have studied transcriptomic and proteomic profiles on samples from 40 B-CLL pts (Binet stage A).

MATERIAL AND METHODS: 100 μg of total PBMC proteins were used for IEF followed by 2D electrophoresis. Image analysis of scanned gels was used to identify statistically differentially expressed proteins. Image acquisition and analysis were performed using the Ludesi software (http://www.ludesi.com). Selected spots were subjected to automatic digestion and the proteins were identified by MALDI-TOF (Voyager DE-Pro, Applied Biosystems) peptide mass fingerprint using the Protein Prospector software. To confirm the initial results, sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with a nESI-QTRAP mass spectrometer from Applied Biosystems. Eight proteins were validated by Western Blot.

Total RNA from B cells was used to analyze the expression profile by hybridization with Whole Human Genome U133 Plus 2.0 Array from Affymetrix. Normalization, differential gene expression and functional annotations were analyzed using the GEPAS suite (http://www.gepas.org). qPCR using TaqMan primers/probes was used for validation of the microarray data

RESULTS: When we compared IgVH mut vs unmut transcriptomic and proteomic profiles, we found more than 600 differentially expressed genes and 12 proteins ( fdr <0.05 adjusted for multiple test contrast and p<0.05, Mann-Whitney’s test, for gene and proteins, respectively). In tables 1 and 2 we show some of the most differentially expressed gene/proteins in each group of pts. Validation of results from microarrays and proteomic data using qPCR and Western blot are in progress. We obtained positive correlation between transcriptomic and proteomic profiles, (corr=0.21, p=0.04, Pearson’s correation test) suggesting that common features are found using both approximations.

CONCLUSION: We found a number of interesting gene/proteins that could be able to differentiate molecular subgroups of B-CLL pts. The study of these proteins and genes may lead to better understand the different clinical behaviour of IgVH mut and unmut B-CLL forms, but validation with a larger group of pts is still necessary.

Table 1: Genes differentially expressed

IgVH mut IgVH unmut Genes annotated using their gene symbol BCL11A MGC9913 DUSP22 RGS4 PDLIM5 CRY1 RDH13 GGT2 PHF15 DMD SVH TUBB6 ADAM29 LPL ITPKB ITGA9 RBKS BCL7A NFATC1 MYEOV RIN3 PPP1R9A Table 2: Proteins differentially expressed IgVH mut IgVH unmut Proteins were annotated according to their gene symbol VIM ERP29 COTL1 CCT2 S100A9 PSMB10 HSPD1
The Effect of Interleukin-11 (Oprelvekin, Neumega®) on Thrombocytopenia Associated with Tyrosine Kinase Inhibitors (TKI) in Patients with Chronic Myeloid Leukemia (CML).
Authors: Aribi Ahmed M. , Kantarjian Hagop , Ault Patricia , Koller Charles Asa , Thomas Deborah , Faderl Stefan , Garcia-Manero Guillermo , Cortes Jorge
Published: October 2019

During therapy with Imatinib and other TKI, 20–50% of patients with CML develop grade ≥3 thrombocytopenia. This frequently leads to treatment interruptions and dose reductions that may decrease the probability of achieving a cytogenic response. IL-11 is a megakaryopoietic cytokine that reduces the incidence and the severity of thrombocytopenia associated with chemotherapy. We investigated the efficacy and the safety of low dose IL-11 in improving thrombocytopenia associated with imatinib and other TKI therapy in 13 patients (pts) with CML. Pts were included if they

had CML in chronic or accelerated phase receiving therapy with imatinib, nilotinib, or dasatinib; developed grade ≥3 thrombocytopenia (platelets <50x109/L) after the first 4 weeks of therapy with TKI, and had thrombocytopenia that was either recurrent or requiring TKI dose reductions.

The starting dose of IL-11 was 10mcg/kg 3 times weekly and the dose escalated by 1 dose level every 2 weeks if the patients had no sustained platelet increase ≥10x109 /L above the baseline on at least 2 consecutive measures 1 weeks apart.

The median age is 52 yrs (range, 34–77 yrs). Median platelet count at diagnosis was 164 x109/L (range, 911–72 x109/L); median platelet count at the time IL-11 was started was 37 x109/L (range, 3–74 x109/L). Eleven pts were in chronic, and 2 in accelerated phase. Eight pts were treated with imatinib with a dose range from 300 to 800 mg/day; 3 pts with dasatinab 70–75 mg twice daily; and 2 pts with nilotinib 400mg twice daily. All pts had dose reduction of the TKI prior to initiation of IL-11. After initiation of IL-11, eight of 13 pts had an increase in platelet count with median peak platelet count of 102 x109/L (range, 51–160 x109/L), including 1 pt that normalized platelet counts. Only one of the 8 responders had further TKI dose reduction after IL-11 was started; one of the 8 responders was taken off study secondary to noncompliance, and 1 had IL-11 therapy discontinued secondary to fatigue. One pt that did not show a platelet increase had however his imatinib dose increased from 300 to 500 mg daily. Seven patients had decrease in the number of days of TKI therapy interruption secondary to thrombocytopenia after initiation of IL-11 (12 % of the total treatment time as compared to 30% before the start of interleukin-11). There was no significant toxicity observed with IL-11 therapy except for grade 4 fatigue in one patient which led to discontinuation of therapy and grade 2 peripheral edema in one patient which led to dose reduction.

We conclude that the IL-11therapy in CML pts with ≥ grade 3 thrombocytopenia may lead to improvement in platelet count and minimize treatment interruptions of TKI. At the doses used in this study, IL-11 can be administered with minimal toxicity in most pts.
The FcR γ Chain Potentiates Outside-In Integrin Signaling.
Authors: Boylan Brian , Newman Debra K. , Newman Peter J.
Published: October 2019

The FcRγ chain is a 12 kDa homodimer that exists in the platelet plasma membrane non-covalently associated with platelet membrane glycoprotein (GP)VI. Interaction of laminin or collagen fibers in the vessel wall with GPVI results in rapid transmission of activation signals into the cell - a process that is dependent on Src family kinase-mediated phosphorylation of Immunoreceptor tyrosine-based activation motifs (ITAMs) within the cytoplasmic domain of FcRγ. Tyrosine phosphorylated FcRγ, in turn, recruits the tyrosine kinase Syk, which then initiates a signal amplification pathway involving assembly of a series of adaptor proteins, activation of phospholipase Cγ2, and generation of second messengers that facilitate granule secretion, activation of the platelet integrins, and platelet aggregation. We and others have recently shown that loss of the GPVI/FcRγ chain complex from the surface of either human or murine platelets renders them unresponsive to collagen and laminin. Unexpectedly, GPVI/FcRγ-chain-depleted platelets also exhibited moderately reduced reactivity to adenosine diphosphate (ADP) and thrombin, which activate platelets by binding to the G protein coupled receptors (GPCRs) P2Y1, P2Y12, PAR1, and PAR4, respectively. Because GPCR-mediated platelet activation has not previously been thought to involve signals emanating from the GPVI/FcRγ-chain complex, we sought to determine whether the FcRγ chain might function to amplify platelet activation responses following GPCR-mediated signaling. Human platelets were exposed to ADP, thrombin or the TXA2-mimetic, U46619, allowed to aggregate, and detergent lysed. Phosphotyrosine immunoblot analysis revealed that the FcRγ chain became rapidly phosphorylated on its ITAMs during platelet aggregation induced by either thrombin or U46619. Interestingly, FcRγ chain phosphorylation was completely inhibited when platelets were activated in the presence of RGD peptides, which allow platelet activation and granule release, but prevent fibrinogen binding to the major platelet integrin, GPIIb-IIIa. The observation that the FcRγ chain becomes tyrosine phosphorylated following GPCR signaling in an RGD-inhibitable manner suggests that integrin-associated kinases might be capable of phosphorylating FcRγ, either directly or indirectly, downstream of ligand binding, though evidence for this remains to be determined. Taken together, these dataprovide support for the notion that the FcRγ chain functions as a general amplifier of platelet activation,helps to explain why GPVI-depleted platelets exhibit blunted responses to agonists specific for GPCRs, andmay lead to important new insights into the mechanism of action of contemplated GPVI-based therapeutics.
Correlations between BCR-ABL Ratio and Cytogenetic Response at Months 12 of the Randomized French SPIRIT Study on Behalf the Fi-LMC Group.
Authors: Preudhomme Claude , Roche-Lestienne Catherine , Crinquette Antoine , Roumier Christophe , Eclache Virginie , Guilhot Joëlle , Guilhot Francois , Mahon François-Xavier
Published: October 2019

In September 2003, the French CML study group (Fi-LMC) activated the four armed randomized SPIRIT trial comparing imatinib 400mg with imatinib 600mg, imatinib 400mg + ara-C and imatinib 400mg + pegylated interferon in newly diagnosed CML in chronic phase. The molecular monitoring was centralized and performed, according to the EAC protocol, in duplicate on the same BCR-ABL cDNA among two French laboratories (Bordeaux and Lille). Data were expressed as BCR-ABL/ABLx100 on the new internationally agreed scale (IS) using a conversion factor of 1.33 and 0.80 for Bordeaux and Lille respectively. A good correlation between the two laboratories was observed. During the first year of follow-up the quantification was performed each three months (M0 to M12). In May 2006, 297 of the 492 enrolled pts had a follow up of more than 12 months, and 263 pts were analysed at M12. Using the IS, the median values of the BCR-ABL/ABL normalized ratios were 90%, 6,4%, 0.68%, 0.32% and 0.16% at M0, M3, M6, M9, and M12 respectively. At M12, 15%, 29%, 27%, 17% and 12% of the pts presented a BCR-ABL/ABL ratio lower than 0.01%, 0.1% ,1%, 10%, and 100% respectively. Overall, 71% of the pts presented a ratio lower than 1%. Conventional cytogenetic data were available for 193 pts at M12: 156 pts (80%) were in complete cytogenetic response (CCR), 25 pts (13%) were in major cytogenetic response (MCR) and 12 pts (6.2%) were in minor or a lack of cytogenetic response. Among pts with a BCR-ABL transcript ratio lower than 1%, all except four were in CCR. The last 4 cases were in MCR associated with BCR-ABL ratios of: 0.96% for one pt, 0.08% and 0.12% for 2 pts (who further reached CCR at 6 and 18 months) and 0.128% for the last one. Percentages of Philadelphia positive metaphase (Ph+) were 4%, 6%, 5 % and 15% respectively. Among the 30 pts with a BCR-ABL ratio > 5%, none were in CCR and only 18 pts were in MCR. Among the 27 pts presenting a BCR-ABL ratio in the 1%–5% intervals, 20 pts were in CCR and 7 pts were in MCR (5 pts with Ph+ lower than 10% and two pts with 16% and 20% Ph+).

In conclusion, our findings suggest that a ROC statistical test will be able to determine critical BCR-ABL/ABL ratios associated to CCR in our study. Nevertheless, at M12 the percentage of available data was superior using molecular than cytogenetic monitoring, suggesting that molecular monitoring expressed with the new international scale is probably sufficient fore more than 85 % of the pts treated by IM solely or in association.
A Half-Log Increase in Quantitative RT-PCR for BCR-ABL (RQ-PCR) as a Trigger for Kinase Domain Mutation Analysis.
Authors: Press Richard D. , Willis Stephanie , Rempfer Carole , Mauro Michael J. , Druker Brian J. , Deininger Michael W.N.
Published: October 2019

RQ-PCR for BCR-ABL is standard of care for monitoring patients with chronic myeloid leukemia on Abl kinase inhibitor therapy, and mutations in the kinase domain (KD) of BCR-ABL are the most frequent mechanism of acquired drug resistance. From a practical point of view, it is important to establish an RQ-PCR trigger for mutation analysis that is sufficiently sensitive, while avoiding a high rate of false positive results. In one study, a single 2-fold (0.3 log) rise in BCR-ABL transcript levels predicted mutations in 61% (1). However, the results were not confirmed in a similar study(2). We therefore analyzed the correlation between a slightly more conservative 0.5 log rise in RQ-PCR levels and mutation detection in an unselected cohort of imatinib-treated CML patients. A database search identified 273 samples (from 150 patients) with a half-log or greater RQ-PCR rise. Mutation analysis (by direct DNA sequencing) has been completed on 54 samples. BCR-ABL levels were higher in 44 samples with successful amplification and sequencing compared to 10 without sufficiently good sequencing quality [median log-drop 0.31 (range −1.2 − 2.4) vs. 2.2 (range 0.17 – 3.2), p<0.0001]. KD mutations were detected in 11 samples (25%) and included L248V, G250E, Y253H, E282G, E292V, T315I (n=2), F359V+H396R, V379I, F382L, F482S). A similar mutation rate was detected by using a trigger of a 1.0-log increase of RQ-PCR levels [4 mutations out of 19 eligible samples (21%)], but with this higher trigger level, 7 patients with mutations would be excluded from detection. We then analyzed the risk of relapse according to mutation detection. The 44 samples with a >0.5 log RQ-PCR increase were from 34 different patients. Eleven of these 34 patients had cytogenetic or hematologic progression at the time of the mutation analysis and 7 never achieved even a partial cytogenetic response (<65%). Of 16 remaining eligible patients, 9 subsequently relapsed at a median of 5.8 (range 2.6–17) months after mutation detection. Freedom from progression was shorter in patients with a half-log RQ-PCR increase compared to patients without (34 months from imatinib start vs. median survival not reached, p = 0.019).

Conclusion: In our hands, a half-log increase of BCR-ABL levels is associated with mutation detection in 25% of samples, yields a significantly increased risk of subsequent relapse, and may be used as a practical trigger for kinase domain sequencing. Analysis of additional samples is ongoing to determine the ideal trigger level that optimizes specificity and sensitivity.
Imatinib Mesylate Discontinuation in Patients with Chronic Myelogenous Leukemia in Complete Molecular Remission: An Update Follow Up.
Authors: Mahon Francois-Xavier , Huguet Francoise , Etienne Gabriel , Réa Delphine , Cayuela Jean-Michel , Marit Gerald , Gardembas Martine , Reiffers Josy , Philippe Rousselot
Published: October 2019

The BCR-ABL tyrosine kinase inhibitor imatinib mesylate (Gleevec) induces complete cytogenetic responses (CCR) in more than 85% of patients with chronic myelogenous leukemia (CML). However, patients in CCR relapse after imatinib interruption in case of detectable residual disease. In fact, less than 10% of patients achieve a molecular remission, defined by an undetectable residual disease using real time quantitative polymerase chain reaction (RTQ-PCR). We previously reported the outcome of CML patients in CCR after cessation of interferon-alpha during the pre-imatinib era. Seven (all with a negative PCR) out of 15 patients did not relapse (J Clin. Oncol.,20,2002:214–220). In the present study, we address the issue of the discontinuation of imatinib in CML with undetectable residual disease for more than 2 years in 15 patients. The median duration of RTQ-PCR negativity and imatinib therapy were respectively 32 months (24–46) and 45 months (32–56) before imatinib interruption. Eight patients displayed a molecular relapse with a detectable BCR-ABL transcript appearance between the first 6 months. Imatinib was then re-introduced and led to a novel molecular response in most patients. Seven other patients have still an undetectable level of BCR-ABL transcript after a median follow up of 20 months (9–24). With the assumption that the doubling time of a proliferative CML cell is 8 days, it will take a maximum of 6 months if only one leukemic cell persists and proliferates to reach 107 cells i.e corresponding to a residual disease detectable by RTQ-PCR. Relapses observed within 6 months may reflect the kinetic of undetectable dividing CML cells. Those cells may be eradicated or controlled in long term non relapsing patients described in our study.
Imatinib Mesylate Therapy in Late Ph+ Chronic Myeloid Leukemia Patients in Stable Complete Cytogenetic Response after Interferon-Alpha Results in a Very High Complete Molecular Response Rate.
Authors: Alimena Giuliana , Breccia Massimo , Luciano Luigia , Quarantelli Fabrizio , Diverio Daniela , Izzo Barbara , De Angelis Biagio , Mancini Marco , Latagliata Roberto , Carmosino Ida , Nanni Mauro , Rotoli Bruno , Mandelli Franco , Pane Fabrizio
Published: October 2019

Imatinib mesylate was given to 26 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients who were in late chronic phase (CP) and in stable complete cytogenetic response (CCR) after interferon-alfa (IFN-α), but showed persistent positive residual disease at PCR analysis under this treatment. At diagnosis median age was 40 years (range 21–64) and according to Sokal’s score, 18 patients were low risk and 8 were intermediate risk. Median IFN treatment was 88 mo.s (range 15–202) and median CCR duration was 73 mo.s (range 10–148). Imatinib was administered at the standard dose of 400 mg/die, after stopping IFN for 1 week. Residual disease was measured on bone marrow (BM) cells at baseline, before starting Imatinib, at 3, 6, 12, 18 mo.s and at the last follow-up (median 32 mo.s, range 21–49), by assaying BCR-ABL transcripts using quantitative PCR (RQ-PCR). The copy number (CN) of BCR/ABL and ABL transcript were derived by the interpolation of CT values to the appropriate standard curve, and the result, for each sample, was expressed as ratio of BCR/ABL mRNA copies to ABL mRNA x 100 (normalized copy number - NCN).

Imatinib treatment resulted in a progressive and consistent decline of residual disease in all but one patient, from a median of 0.89 at baseline to 0.01 at the end of follow-up. Major molecular response (BCR/ABL levels <0.1) was reached in 20 patients (77%) and BCR/ABL transcripts were undetectable in 13 (50%). Achievement of molecular response was significantly correlated with post-IFN baseline transcript level (mean 1.194 for patients achieving complete molecular response vs 18,97 for those who did not; p<0.001), but not with other clinical/biological patient characteristics. In all patients, imatinib was well tolerated with no side effects requiring drug dose reduction or dose discontinuation.

Albeit obtained from an unusual subset of selected patients with favourable prognosis, and likely particularly sensitive to imatinib, present results confirm the efficacy of combining Imatinib and IFN-α and further support investigating treatment approaches employing these two drugs.
Dasatinib (SPRYCEL®) in Patients (pts) with Chronic Myelogenous Leukemia in Accelerated Phase (AP-CML) That Is Imatinib-Resistant (im-r) or -Intolerant (im-i): Updated Results of the CA180–005 ’START-A’ Phase II Study.
Authors: Cortes Jorge , Kim D.W. , Guilhot F. , Rosti G. , Silver R.T. , Gollerkeri A. , Agarwal P. , Branford S. , Apperley J.F.
Published: October 2019

Dasatinib (SPRYCEL®, formerly BMS-354825) is a novel, oral, multi-targeted kinase inhibitor that induces complete hematologic and cytogenetic remissions in pts in all phases of im-r or im-i CML. START-A is an open-label study of dasatinib in pts with im-r or im-i AP-CML. Preliminary results with early follow-up suggested significant activity. The present report updates the results of this study with a minimum of 9 months of follow-up. Dasatinib was given orally at 70 mg twice daily (BID). Dose escalation to 100 mg BID was allowed for inadequate initial response and reduction to 50 or 40 mg BID for persistent toxicity.

Evaluation included weekly blood counts and monthly bone marrow including cytogenetics. Molecular evaluation of BCR-ABL transcript levels by real-time qPCR was performed at baseline and upon documentation of complete cytogenetic response. A total of 174 pts (161 im-r and 13 im-i) were enrolled between December 2004 and July 2005 in 39 centers worldwide. There were 96 (55%) males; median age was 57 years (range 22–86); median time from original diagnosis of CML was 82 months.

Prior therapy included im in all pts (>600 mg/day in 91 (52%), im for >3 years in 103 (59%) pts, interferon in 126 (72%) pts, stem cell transplantation in 23 (13%) pts. Major cytogenetic response (MCyR) to prior im had been seen in 57 (33%) pts. The average daily dose (median across all pts) was 130 mg/day (range 44–199). Major hematologic response (MaHR) was documented in 110 (63%) pts with complete hematologic response in 75 (43%) and no evidence of leukemia in 35 (20%). At 9 months, 85% of pts have maintained their MaHR. MCyR was documented in 65 (37%) pts, complete in 49 (28%), partial in 16 (9%). Median progression-free survival (PFS) has not been reached; estimated PFS at 9 months is 70%. In the 94 pts with BCR-ABL mutations at baseline the MaHR rate was 69%. Generally, response rates were similar in the im-i and im-r groups. Cytopenias were significant with grade 3–4 thrombocytopenia and neutropenia in 82% and 76% of pts, respectively. Non-hematologic toxicities were generally mild to moderate. The most frequent (% any grade, % grade 3–4) were diarrhea (51%, 8%), headache (29%, 1%), fatigue (26%, 4%), fever (25%, 3%) and pleural effusion (25%, 3%). Pleural effusions were optimally managed with dose interruption and diuretics and/or pulse steroids. Dasatinib is highly effective in pts with im-r or im-i AP-CML with high rates of durable MaHR and MCyR. An updated analysis with at least 12 months of follow-up, including molecular response data and mutational analysis at time of progression, will be presented.
Treatment with Nilotinib for Patients with Chronic Myeloid Leukemia (CML) Who Failed Prior Therapy with Imatinib and Dasatinib.
Authors: Jabbour Elias , Kantarjian Hagop , Giles Francis , O’Brien Susan , Cortes Jorge
Published: October 2019

Nilotinib (AMN107) is a selective Bcr-Abl kinase inhibitor which is aproximately 30-fold more potent than imatinib. Phase I and II studies confirmed the efficacy of nilotinib in imatinib-resistant CML. Dasatinib (BMS-354825) is a dual Src-Abl kinase inhibitor recently approved for the treatment of CML in all stages of the disease after imatinib failure. With the availability of more new tyrosine kinase inhibitors, one important question is the existence of cross-resistance between these new agents. To help answer this question, we analyzed the outcome of patients with CML who receive therapy with nilotinib after having failed therapy with dasatinib. Seven patients were treated: 6 had previously shown hematologic resistance to imatinib and one had intolerance to imatinib. Dasatinib failure was due to hematologic resistance in all 7 patients after a median of 16 weeks (range 4 to 32) on therapy. Median age was 50 years (range, 15 to 78 years); median follow-up on nilotinib was 5 months (range, 1 to 14 months). At the start of therapy with nilotinib, 1 patient was in chronic (CP), 1 in accelerated (AP), and 5 in blastic phase (BP). Mutations were found in 2 of 4 patients assessed before nilotinib therapy (F317L and E355G, respectively). All patients received nilotinib 400 mg BID (n=5) or 800 mg single daily dose (n=2). Of the 7 evaluable patients, one patient (in AP) responded (complete hematologic response). This patient had no mutations identified at baseline. At the last follow-up, 5 patients are alive: 3 with active disease, one in CHR, and one in complete molecular remission post allogeneic stem cell transplantation. We conclude that nilotinib has modest activity in patients with CML resistant to both imatinib and dasatinib.
IRF-4 Functions as a Myeloid Tumor Suppressor.
Authors: Acquaviva Jaime , Schatz Jonathan , Singh Harinder , Ren Ruibao
Published: October 2019

Interferon regulatory factor 4 (IRF-4, also know as pip, LSIRF, ICSAT and MUM1) is a transcription factor with well-characterized functions in B and T cell development and immune response regulation. Though its expression has been demonstrated in macrophages, its function in the myeloid system is not as well characterized. The closely related IRF family member IRF-8 (ICSBP) has more clearly defined functions in the myeloid system. IRF-8 is a critical regulator of myelopoiesis, and its loss has been implicated in the pathogenesis of chronic myelogenous leukemia (CML). IRF-8 deficient mice manifest a CML-like syndrome, and we have shown previously that forced expression of IRF-8 in a BCR/ABL-induced murine model of CML represses the resulting myeloproliferative disease and prolongs survival. In the B-cell system, we have found that IRF-4 and IRF-8 function redundantly at the pre-B-to-B transition point of development. In this study we investigate whether IRF-4 and IRF-8 may also have overlapping function in myeloid cells. Our results show that mice deficient in both IRF-4 and IRF-8 develop from a very early age a more aggressive CML-like disease than mice deficient in IRF-8 alone. Bone marrow progenitors from IRF-4/8 DKO mice have increased proliferation, GM-CSF stimulated granulocytic differentiation, and colony-forming capacity compared to progenitors from WT or single KO mice. These data suggest that IRF-4 plays a role in myeloid lineage development and may act to suppress proliferation and granulocytic differentiation of myeloid progenitor cells. We further examined if, like IRF-8, IRF-4 is able to suppress BCR/ABL mediated transformation. We used a murine stem cell virus vector to co-express BCR/ABL and either IRF-4 or IRF-8 in BM progenitor cells and assessed colony forming capacity of infected cultures as well as the ability of IRF-4 to suppress BCR/ABL induced CML-like disease in a murine model. Our results demonstrate that IRF-4 potently suppresses BCR/ABL induced colony formation and that forced expression of IRF-4 suppresses BCR/ABL-induced CML-like disease in mice even more potently than IRF-8. These results provide direct evidence that IRF-4 is an important myeloid tumor suppressor and may allow elucidation of new molecular pathways significant to the pathogenesis of human CML.
Leukemic Transformation of Primary Human CD34+ Progenitor Cells by MLL-AF9 Recapitulates the Phenotype and Gene Expression Profile of MLL Rearranged AML.
Authors: Mulloy James C. , Wei Junping , Rettig Catherine , Wunderlich Mark , Alvarez Sara , Cigudosa Juan , Jansen Michael , DiMartino Jorge F.
Published: October 2019

The t(9;11)(p22;q23) results in the creation of an MLL-AF9 fusion gene and is observed in 27% of 11q23 leukemias. t(9;11) is commonly associated with an M5 monocytic leukemia and patients harboring this translocation are included in the intermediate or poor cytogenetic risk groups, with an average 42% overall survival at five years. Using primary human hematopoietic progenitor cells, we have developed a system that closely mimics the MLL-AF9 associated disease observed in human acute myeloid leukemia (AML). Injection of MLL-AF9 expressing cells into sublethally-irradiated NOD/SCID mice produced an aggressive FAB-M5-like leukemia that infiltrated the spleen and liver and induced death with an average latency of 8 weeks. Serial transplantation of leukemic cells recapitulated the disease with a similar latency. MLL-AF9 expression conferred the ability to grow indefinitely in culture, promoted an accumulation of cells resembling monoblasts and was associated with an increase in telomerase activity. Mono- or oligoclonal outgrowth arose in vitro as well as in vivo. Numeric karyotypic changes or normal karyotypes, in the absence of structural rearrangements, additions or deletions were detected in cultured cells as well as samples from leukemic mice. Principal Component Analysis of genome-wide gene expression data from MLL-AF9 expressing cultured cells revealed a gene expression signature that closely mirrors that observed in MLL-rearranged leukemic cells from patient samples. Furthermore, training of a (linear) Support Vector Machine with gene expression data from patient samples resulted in the correct classification of all MLL-AF9-expressing cell cultures as MLL-rearranged leukemia. We conclude that expression of MLL-AF9 in a normal human hematopoietic progenitor is sufficient to recapitulate many key aspects of the clinical disease. This model will yield valuable insights into the molecular pathogenesis of MLL fusion driven leukemia and will serve as a powerful in vivo model for testing much needed novel therapeutic targets.
The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.
Authors: Pulikkan J. , Zada A. Peer , Geletu M. , Dengler V. , Tenen Daniel G. , Behre G.
Published: October 2019

CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.
Hypermethylation of FANCC and FANCL Resulting in a Mitomycine-C (MMC) Sensitive Cellular Phenotype in Sporadic Acute Leukemia.
Authors: Hess Corine J. , Ameziane Najim , Schuurhuis Gerrit Jan , Errami Abdellatif , Denkers Fedor , Hubeek Isabelle , Kaspers Gertjan J.L. , Joenje Hans , Reinhardt Dirk , Ossenkoppele Gert J. , Zwaan Christian M. , Waisfisz Quinten
Published: October 2019

The proteins of the Fanconi anemia/BRCA pathway are thought to function in specific DNA repair processes. Disruption of these genes results in genomic instability and hypersensitivity to DNA cross-linking agents. As illustrated by patients with Fanconi anemia (FA) [OMIM#227650] this cellular phenotype confers an increased risk for the development of malignancies, in particular acute myeloid leukemia (AML). In the past years several studies have shown that inherited and somatic abnormalities in individual FA genes play a role in the development of sporadic cancers. To study the involvement of the FA genes in sporadic cases of leukemia we analyzed the presence of aberrant promoter methylation of the 11 known FA genes by Methylation Specific-Multiplex Ligation dependent Probe Amplification (MS-MLPA).

We studied 119 unselected adult AML bone marrow samples and 20 pediatric AML samples that were selected on the bases of a complex karyotype. A single adult patient sample, diagnosed biphenotypic AML, showed aberrant methylation of the promoter region of the FANCC gene. The aberrant methylation pattern was also present at relapse. Another four biphenotypic AML samples, together with 15 adult and 82 pediatric ALL samples were subsequently analyzed. In this group, one adult ALL sample showed FANCL promoter methylation and three pediatric ALL samples showed FANCC promoter methylation. Phenotypically all four ALL samples were classified as hyperdiploid B-cell precursor ALL. Sodium bisulphite sequencing confirmed hypermethylation of the respective genes in all cases. Colony Forming Unit (CFU) assays were performed to analyze whether hypermethylation of FANCC and FANCL result in an FA-like phenotype of DNA cross-linker sensitivity. Colony formation in the methylated samples as well as control leukemic samples, i.e. without FA-gene methylation, was scored after proliferation in the absence or presence of MMC. Two pediatric ALL samples with FANCC hypermethylation did not show colony formation in the CFU assay. The other four samples with FA gene methylation, including the relapse sample from the biphenotypic AML patient, demonstrated hypersensitivity to MMC in a CFU assay when compared to the controls.

In summary, this is the first report showing FANCC and FANCL promoter hypermethylation in acute leukemia with either partial or complete lymphoid differentiation. Silencing of the corresponding FA genes may contribute to the leukemogenic process by imposing an increased level of spontaneous genomic instability. Cross-linkers may thus be of therapeutic use in these selected patients.
DNA Methylation Independent Silencing of the RARα Gene Expression in Acute Myeloid Leukemia.
Authors: Glasow Annegret , Barrett Angela , Chornet Manuel Boix , Gupta Rajeev , Zhou Da-cheng , Gallagher Robert , von Lindern Marieke , Waxman Samuel , Enver Tariq , Hildebrandt Guido , Zelent Arthur
Published: October 2019

All-trans-retinoic acid (ATRA) and the gene encoding retinoic acid receptor-α (RARα) have been implicated in the pathogenesis and treatment of acute promyelocytic leukemia (APL). Nevertheless, the role of these molecules in the pathogenesis and therapy of non-APL acute myeloid leukemias (AMLs) remains unclear. Previously we have shown that expression of the ATRA-inducible RARα2 isoform is downregulated in a variety of AML cell lines and increases with hematopoietic differentiation along the myelomonocytic lineage. Using quantitative real-time PCR we have now investigated expression of the RARα gene in primary AML cells (n=23) and report that as in AML cell lines the levels of RARα2 mRNAs are markedly reduced (by 48 fold in APL, p≤0.05, and by 52 fold in non-APL AML, p≤0.01) relative to cord blood (CB) derived CD33 (or CD34) positive cell population (n=4). However, in contrast to the AML cell lines and normal hematopoietic progenitors, the expression of the RARα1 isoform was also significantly reduced in primary AML samples (up to 26 fold, p≤0.05). Examination of potential mechanisms underlying the silencing of the RARα gene expression in AML revealed that the RARα2 promoter possesses two small CpG islands that are fully methylated in all AML cell lines examined. Consistent with the expression pattern of RARα1 a single CpG island in the RARα1 promoter region was unmethylated in all these samples. As expected from such results expression of RARα2, but not RARα1, could be stimulated with a DNA demethylating agent 5-aza-2′deoxycytidine and synergistic effects between 5-aza-2′deoxycytidine and ATRA were observed on both RARα2 expression and cellular differentiation of APL and non-APL AML cell lines. Extending this analysis to clinical material we have surprisingly discovered that RARα1 and RARα2 CpG islands are unmethylated in all AML patient samples, including 3 APL cases, suggesting that DNA methylation may not play a significant role in silencing of the RARα gene expression in primary AML cells. Chromatin immunoprecipitation of the RARα2 regulatory region with antibodies to specific histone modifications revealed presence of other negatively acting chromatin states in primary AML samples (relative to normal CD33 positive CB cells), including decreased histone H3 acetylation as well as decreased di- and tri-methylation of histone H3 lysine 4. A possibility that microRNAs targeting sequences in the common 3′-UTR regions of the RARα1 and α2 isoforms may also contribute to silencing of both RARα1 and α2 expression in AML cannot be excluded. These results underscore the complexities of mechanisms that are responsible for silencing of gene expression in AML and support the notion that diminished RARα expression contributes to leukemogenesis.
Identification of Oncogenic Pathways of T-Acute Lymphoblastic Leukemia (T-ALL) through Gene Expression Profiling of Mouse Tumor Models.
Authors: Sulis Maria Luisa , Palomero Teresa , Real Pedro J. , Barnes Kelly C. , Aplan Peter , Copeland Neal , Utpal Dave , Gounari Fotini , Grosveld Gerald , Kappes Dietmar J. , Kee Barbara , Kelliher Michelle , Lenz Jack , Richardson Christine , Ferrando Adolfo
Published: October 2019

Transgenic mouse models of T-cell oncogenes such as TAL1 and LMO1, have confirmed their critical role in the development of T-lymphoblastic lymphoma (T-LL). In addition, retroviral insertional mutagenesis (RIM) in mice is a powerful system for the identification of genes involved in T-cells leukemogenesis. However, incomplete knowledge of the pathogenesis of T-ALL limits the ability to stratify patients and to deliver tailored therapy accordingly. Thus, a major goal is to identify common oncogenic pathways downstream of T-cell oncogenes that can serve as therapeutic targets for the treatment of T-ALL. We hypothesize that T-cell oncogenes operate through a limited number of oncogenic pathways with distinct gene expression profiles. Furthermore, we propose that gene expression profiling of murine models of T-cell lymphomas, which harbor specific genetic lesions, will serve to identify such oncogenic pathways and to establish a molecular classification of T-ALL. To test this hypothesis we have analyzed normal thymus and mouse T-cell lymphomas originated from retroviral insertional mutagenesis (n=11) and transgenic and knock-outs models (n=30) using Affymetrix 430A2.0 microarrays. Unsupervised analysis clearly distinguishes tumor samples from normal thymus and clusters the tumors into three major groups. TAL1, LMO2 and E2 proteins are known to be part of a transcriptional complex and to cooperate in T-cell leukemogenesis by suppressing E2A function. Accordingly, mouse tumors originating in TAL1, TAL1/LMO2 transgenic and E2A knock-out mice share a common gene expression signature and cluster together. Supervised analysis of tumors generated by retroviral mutagenesis showed increased expression of the proviral tagged genes and identified corresponding known downstream targets. Nearest neighbor analysis identified high levels of Notch1 expression in tumors with proviral insertion in the Notch1 locus and in tumors generated in the TAL1/, TAL1/LMO2, OLIG2/LMO1, ThPOK and Ikaros mouse models, which harbored activating mutations in NOTCH1. Our results demonstrate that gene expression profiling identifies common oncogenic pathways in T-cell tumors generated in mice, establishes common mechanisms of transformation for several T-ALL oncogenes and allows coupling of poorly characterized genes identified in proviral insertional sites with well characterized oncogenes and downstream molecular pathways. The identification of mechanisms of T-cell transformation common to tumors of different origin lays the ground for the identification of new therapeutic targets for the treatment of T-ALL.
Identification of Normal and Neoplastic Human Mature B Cells “miRome”.
Authors: Basso Katia , Morozov Pavel , Califano Andrea , Rosolen Angelo , Chau Jacqueline , Chen Chang-Zheng , Dalla-Favera Riccardo
Published: October 2019

The recent discovery of microRNAs (miRs) has added a new dimension to the mechanisms that regulate gene expression in normal cell development. Initial evidence also suggests that structural or functional alterations of miRs are associated with tumorigenesis. Nonetheless, the full set of genomic miRs is not known for any eukaryotic species including humans, since presently known miRs have been identified by virtue of their expression in only a few cell types. Analogous to other transcriptional units, it is conceivable that a significant part of the entire miR genomic pool may display tissue-specific expression. In order to understand the role of miRs in mature B cell function and lymphomagenesis, we planned the isolation of the full set of miRs (miRome) expressed in germinal center (GC) B cells and in GC-derived B cell lymphomas. Toward this end, we constructed libraries representative of all miRs expressed in normal GC B cells isolated from human tonsils and in a Burkitt lymphoma cell line (Ramos). Short RNAs were gel purified and linked to adaptor oligonucleotides, reverse transcribed, PCR amplified, cloned into an expression vector, and subjected to sequencing. Candidate miRs have been aligned to the human genome to retrieve their potential precursor sequences, whose folding characteristics have been analyzed in order to identify bona fide precursors. The cloned and structurally validated miR precursors have been matched to the public miR database to detect previously identified miR sequences. Initial results show that 453 miRs cloned from GC B cells correspond to 99 precursors (59 previously known and 40 newly discovered), while 233 miRs cloned from Ramos cell line identified 42 known and 46 new precursors. Overall, this initial analysis led to the discovery of 76 not previously reported miR precursors. Since less than a third of the available clones have been analyzed, it is expected that a significantly larger number of novel miRs will be identified by our method. A comprehensive analysis of miR expression profiles of normal versus neoplastic GC B cells will be obtained using a dedicated miR chip. Interestingly, initial results indicate that a set of miRs is expressed in normal GC cells, but not in Ramos and other GC-derived Burkitt lymphoma cell lines, suggesting that structural and/or functional alterations of miRs occur during lymphomagenesis.
HOX Expression Patterns Identify Major Cytogenetic Groups and Are of Prognostic Importance in Adult AML.
Authors: Andreeff Michael , Ruvolo Vivian , Gadgil Sharvari , Chen Wenjing , Coombes Kevin , Zeng Chan , Baron Anna , Drabkin Harry
Published: October 2019

Naturally occurring and experimentally-induced deregulation of HOX genes has been causally linked to leukemia and myeloproliferative states. We developed and validated SYBR-green based real-time qRT-PCR assays for the clustered HOXA-D genes as well as other selected homeodomain, trithorax and polycomb genes. These were used to examine 120 cases of adult AML (median age 58 y) with the following cytogenetic classifications: favorable (FAV) 17, intermediate (INT) 53, unfavorable (UNFAV) 46, no analyzable metaphases 4, plus normal CD34+ sorted cells from 4 bone marrow donors. HOX expression patterns were intimately linked to the major cytogenetic subsets and provide a unifying feature for some AML subsets, particularly within the favorable prognostic group. Twenty genes were identified, with a 95% confidence of no false positives, that distinguished the major cytogenetic groups. In general, levels of HOXA genes and MEIS1 distinguished FAV vs. INT and FAV vs. UNFAV groups, while the HOXB and PBX3 levels distinguished either all three groups or INT vs. UNFAV. Comparing 68 patients who achieved a complete response with 49 patients who had resistant disease (or failed induction therapy), 11 genes were differentially expressed with parametric p-values < 0.05. Based on permutation distribution with a 95% confidence level, there were no more than 5 false positives among these 11 genes. Using a COX proportional hazards model and patients for whom survival data were available, HOXA9 and HOXA10 were predictive of overall survival, with parametric p-values <0.05. Lastly, compared to normal CD34+ cells, homeodomain gene expression was abnormal in nearly every AML. Based on these results and the role of HOX deregulation in experimental leukemogenesis, we propose that differences in HOX expression may be responsible for some of the biological differences observed among major cytogenetic subsets in human AML.
The mTOR Inhibitor Temsirolimus (CCI-779) Induces Cell Cycle Arrest and Autophagy in Hodgkin Lymphoma (HL) Cell Lines and Enhances the Effect of the PI3-Kinase Inhibitor LY294002.
Authors: Georgakis Georgios V. , Yazbeck Victor Y. , Li Yang , Younes Anas
Published: October 2019

The PI3K/AKT/mTOR pathway is frequently activated in a variety of cancers giving hope that that this pathway may prove to be a good target for cancer therapy. We and others have recently reported that primary and cultured Hodgkin and Reed-Sternberg cells of HL express the active phosphorylated form of Akt (pAkt) which contribute to their survival. Furthermore, we have demonstrated that inhibition of PI3K by the small molecule LY294002 or Akt inhibition induced cell cycle arrest and apoptosis. However, at the present time there is no PI3K or Akt inhibitors in clinical trials. Instead, several inhibitors of the downstream mTOR kinase are already being evaluated in patients with cancer. Temsirolimus (CCI-779) is an mTOR inhibitor that demonstrated promising clinical activity in patients with relapsed mantle cell lymphoma. To explore the potential clinical activity of Temsirolimus in HL, we examined its efficacy in HL-derived cell lines (HD-LM2, L-428, and KM-H2). Here, we demonstrate that CCI-779 inhibited mTOR, as evident by the inhibition of ribosomal S6 phosphorylation as early as 6 hours, with a dose as low as 1 nM. Temsirolimus induced antiproliferative cytostatic effect in the same dose (1nM) in all HL cell lines, and this effect did not improve with higher concentrations (up to 1000 nM). Furthermore, the effect was predominantly cytostatic with little or no induction of apoptosis. This antiproliferative effect was predominantly due to cell cycle arrest in G0/G1 phase, and was associated with upregulation of p27 cell cycle protein. Although temsirolimus did not induce apoptosis, it induced autophagy, as determined by Acridine Orange staining of the acidic autophagic intracellular organelles and FACS analysis. Combination of 1nM CCI-779 with increasing concentrations of the PI3K inhibitor LY294002 showed a profound decrease in the IC50 of LY294002 in all 3 cell lines. Moreover, the combination of CCI-779 and LY294002 blocked cell cycle at G0/G1 phase more efficiently and significantly increased autophagic cells when compared with each agent alone, but failed to induce apoptosis. This effect was mediated in part by inhibiting a negative feedback loop involving AKT phosphorylation. Collectively, our data suggest that temsirolimus may have a therapeutic value in HL.