Università di Pisa
Scuola di Specializzazione in Patologia Clinica e Biochimica Clinca
Nuovo Ordinamento
Direttore Prof. Aldo Paolicchi
Effects of replacement of miRNA-15 and miRNA-16 expression in
Chronic Lymphocytic Leukemia
Candidata Relatori
Monica Colombo Prof.ssa Maria Adelaide Pronzato Prof. Aldo Paolicchi
Author statement
Please consider that results here reported have been recently published in a revised form on Leukemia. 2017 Feb 3. doi: 10.1038/leu.2016.394. [Epub ahead of print].
The author of thesis also is co-author of the paper. Text, figure and tables of the thesis are for academic purpose and are different from the revised version published in Leukemia. Acknowledgment: I would like to thanks all of the authors involved in this very big and difficult project. A particular thank to Dr. Giovanna Cutrona for having believed in the project till the end.
ABSTRACT
B-cell chronic lymphocytic leukemia (CLL) is the most common adult human Leukemia. The pathogenetic mechanisms involved in development of CLL are unknown. Several studies demonstrated the central role of deletion of the region 13q14.3 involving microRNA cluster mir15a and mir16-1 that downregulated in more than 60% of the patients. In this study, we
investigated the effect of the replacement of microRNA 15 and 16 in CLL cells with del13q14 by transfection of miRNA mimics into CLL cells in vitro and in a NOD/Shi-scid,γcnull(NGS) mice in vivo. Administration of miRNA mimics determined a decrease of CLL cells viability in vitro, with exception of certain CLL bearing TP53 mutated. Transfection of miRNA mimics in NGS mice, previously injected with CLL cells, resulted in substantial tumor regression. In contrast, transfection of miRNA inhibitors in CLL cells without del13q14 and variable expression of miR15 and miR16, lead to an increase of cells viability in vitro and an enhanced capacity of CLL cells to growth in vivo. These data demonstrate the capacity of the mir15-16 cluster to control clonal expansion providing indications for a target therapeutic strategy based on microRNAs.
INTRODUCTION
CHRONIC LYMPHOCYTIC LEUKEMIA
Background
Chronic Lymphocytic Leukemia (CLL) is one of the most diffuse adult Leukemia in the world.(1). According WHO classification, it is characterized by accumulation of small mature B lymphocytes in the blood, marrow and lymphoid tissues. CLL B cells typically express CD19, CD5 and CD23 and low levels of surface immunoglobulin(2). CLL also express low levels of surface- membrane immunoglobulin and a slight light chain(3). Despite of the extensive efforts, the pathogenetic events leading to initiation and progression of this disease have not been defined(2).
Most often patients are asymptomatic at time of diagnosis and became aware of disease following detection of lymphocytosis in the routine blood count. The initial diagnosis require detection of >= 5000 cells for ul of clonal B CLL. These lymphocytes show great longevity due the inhibition of apoptosis(4).
The usual symptoms of CLL patients are weight loss, fatigue, excessive night sweets, increased frequency infections, anaemia.
The clinical course of the disease is extremely variable, some patients are free of symptoms and fully active for decades, while other rapid become symptomatic and require therapy. About 50% of cases will evolve aggressively.
Many patients however show an intermediate clinical course, between the two extremes above described. Thus, the investigation and assessment of new prognostic factors at time of diagnosis could contribute to identify patients who require therapy soon after
diagnosis and help to define which of the therapy should be applied for a particular group of patients.
Prognostic factor
Several prognostic factors predicting the clinical course have been identified including genetic subgroups, immunoglobulin mutational status, ZAP-70 and CD38 (1, 5-8). Other prognostic factors include age, gender, clinical stage (Rai or Binet), lymphocyte doubling time (LDT), peripheral blood count, BM histology, B2-microglobulin and TP53 mutations. The status of immunoglobulin reflect the stage of normal B cell differentiation from which they origin (6, 7). CLL with unmutated IGHV gene are supposed to derive from cells that did not enter the germinal center, which are the sites in lymph nodes where normal B cells accumulate somatic mutations on their IGHV gene. Unmutated CLL (U-CLL) typically show a more aggressive disease compared to patients with mutated IGHV gene. The mutated CLL (M-CLL) are supposed to derive from post germinal center B cells and show somatic mutations and isotype class switch recombination thus having a behavior similar to normal B cells when they are responding to Antigens (9 ).
The repertoire of BcR of CLL patients is biased with 30% of patients expressing “stereotype” immunoglobulin which are stretches of primary structure in the IGH VDJ rearrangement that can also be identified in the immunoglobulin produced by the CLL of other patients(10). The finding that about 1/75 of CLL patients share the same BcR provide evidence that B-CLL are selected on the basis of the binding capacity of their surface immunoglobulin, thus indicating that BcR plays a crucial role in CLL development. It’s of note that CLL expressing stereotyped BcR IGs can be categorized into different subsets, each of them displaying homogenous biological characteristics, clinical behavior and outcome(11, 12). These studies seem to indicate a role of antigen in CLL ontogeny. However, the nature of molecular antigens involved in leukemia selection is still debated
and an alternative model of BcR stimulation like as the autonomous recognition of a single BcR epitope conserved in all cases has been proposed(13). This model was subsequently modified in order to better explain the biological and molecular differences existing among CLLs, suggesting that BcR of indolent and aggressive CLL interact homotypycally via their combining sites, binding to distinct internal epitope in each subset of CLL(14).
The identification of new prognostic factor, a part of clinical staging, represents one of the most important advance in the understanding pathogenesis. Along the immunoglobulin mutation analysis, genomic alterations are important for classification of the CLL patients (15). Genomic abnormalities are obtained by fluorescence in situ hybridization (FISH) in more than 80% of patients and they include 13q, 11q, 17p, and 6q deletions and trisomy 12 (+12) (15). Del13q has been found in about 50% of the patients (15, 16) including monoclonal B cell lymphocytosis or MBL(17, 18), suggesting a pathogenetic role. Del of 11q and 17p are around 10-15%, Tris12 is around 7-10% and del6q 5-7%. Therefore, these lesions are unlikely to contribute to the initial pathogenetic mechanisms, although they may be involved in both disease progression and resistance to therapy(15, 19-24). Cytogenetic abnormalities can be used to discriminate patients into subgroups with different score risks: low-risk (absence or 13q); intermediate risk (11q, Tris12, 6q); high-risk (17p)(25). However, since patients show different and sometimes discordant prognostic factors, new parameters are needed for a good patient management. Besides, the mutations of certain genes like as NOTCH1, SF3B1, TP53, BIRC3 are enriched in clinically aggressive cases(26).
More recently, different groups focused their interest to microRNAs, that that can be helpful to understand different stages of CLLs.
MicroRNA
evolutionary conserved and involved into regulation of the expression of several genes c(27). The regulation of gene expression occurs mainly through the specific binding of miRNAs to the 3’-un-translated region (3’-UTR) of the messenger RNA of the target gene via a RNA-induced silencing complex(28), although additional mechanisms have been described(29). microRNA are made via a two step processing mechanism for ma primary transcript(pri-miRNA) through an intermediate 60-90 nucleotide length(pre-miRNA) to a final mature microRNA. Dicer and Argonaute family members are required form miRNA precursor processing reaction (30). MicroRNA are able to block the translation on target mRNA by mechanism that are not fully characterized, so inducing a reducing expression of the protein.
MicroRNA expression profile can be used to distinguish normal form malignant B-CLL cells and to differentiate aggressive from indolent disease. (31). Notably in B-CLL, many researchers focused on the most deregulated microRNA, among which we can found mir15-16, mir181, miRs-34b/c.
MicroRNA 15a-16-1
In CLL, biallelic del(13)(q14) results in an incapacity of the cell to express miR15 and miR16 (ref. (32-36)and Supplementary Figure 1) and the deregulation of several target genes, including those involved in cell cycle progression and apoptosis(37-39). This confers an increased resistance to apoptosis and a propensity to leukemic cell proliferation. Low levels of miR15 or miR16 are observed in patients with monoallelic deletions and in many patients without del(13)(q14) (ref. (33, 34, 36, 40-42)). Additional support for the role of the miR15/miR16 locus in CLL pathogenesis comes from the New Zealand Black (NZB) mice strain harboring a germ-line point mutation downstream of the miR16 locus, which prevents normal expression of both miR15 and miR16 and facilitates
leukemia onset and possibly autoimmune manifestations(43). An analogous lesion, present in particular families, genetically predisposes humans to CLL and possibly to other neoplasias(44). Finally, the selective deletion of the miR15/miR16 locus in mice predisposes the development of a CLL-like leukemia(45, 46). Therefore, impairment of miR15 and miR16 function may be involved in promoting the initial phases of leukemogenesis; however, little is known regarding the role of these lesions in maintaining the transformed status and the clonal expansion of full-blown leukemia(47). These first studies were followed by others suggesting that miRNA could contribute to a variable course in CLL. Several of miRNA de-regulated in CLL were related to protein involved in cycle and apoptosis such as BLC2 (MiR15 and miR16)(38), MCL1 (mir29)(48) or TCL1 (miR29 , miR181)(49). The most unfavorable CLL subtype with aberration in TP53 tumor- suppressor gene has also a specific deregulation of certain miRNAs such as miR34 and miR29. The CLL is in this sense a very interesting example of how non coding RNA can be responsible for the onset of the disease or they can be involved in the progression and aggressiveness.
In this study our interest focused on studying the effect of transfection of both miR15 or miR16 in CLL cells in vivo and in vitro studies.
METHODS
Patients and CLL cell preparations.
CLL patients were enrolled within 12 months from diagnosis in the O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Each patient was studied for phenotype, mutational analysis of IGHV gene and the major cytogenetic features as reported . (18, 50, 51).
PBMCs from patients with CLL were isolated by Ficoll-Hypaque (Seromed, Biochrom) density gradient centrifugation. In selected experiments, CD19-positive CLL cells were enriched by negative selection with the EasySep-Human B-cell Enrichment Kit without CD43 depletion (STEMCELL Technologies, Voden Medical Instruments S.p.A).
miRNAs Cell transfection
MirVanaTM miRNA mimics or inhibitors (Ambion Inc, Thermo Fisher Scientific, Grand Island, NY, USA) were transfected into CLL cells by using a Neon Transfection System (Invitrogen, Thermo Fisher Scientific) at the final concentration of 50 nM.
Briefly, 2×106 CLL cells/mL/well were seeded in 24-well plates containing 500 µL of culture medium without antibiotics [RPMI-1640 with L-glutamine and 10% FBS (Gibco, Thermo Fisher Scientific), Sodium piruvate 0.1% (Euroclone)] at 37°C in a 5% CO2 atmosphere. Hsa-miR15a-5p, hsa-miR16-5p, and miRNA Negative Control (CTR)#1, miRNA Inhibitor Negative CTR #1 were employed.
Smartflare and RT-qPCR
MiR15 and miR16 expression was evaluated with two methods: SmartFlare RNA Detection Probes (Merck Millipore, France) in n-13 cases and quantitative real time PCR
(q-RT PCR; n-38 cases).
Smartflare probes were conjugated with the Cyanine 5 (Cy5) fluorophore and were specific for miR15a-5p (miR15 CY5); miR16-5p (miR16 CY5). SF-102 was used as Scramble CTR Cy5. Cells were incubated with Smartflare probes overnight, harvested and analyzed by FACSCanto (BD Biosciences) and DIVA 6 (BD Biosciences) or FLOWJO V.9.8.3 software (Treestar Inc.).
In order to evaluate cell viability, cells were counter stained with propidium iodide (PI; 50 mg/mL, Sigma) in isotonic solution.
The expression of miRNA was calculated as % fold induction or % fold inhibition (see (52) for details).
For real-time PCR, RNA was extracted from cultured cells using the miRVana RNA isolation kit according to the manufacturer's instructions (Ambion-Thermofisher). TaqMan miRNA assays (Applied Biosystems) were performed using a Real-Time PCR system Rotorgene 6000 (QIAGEN) miRNA levels were normalized to the level obtained for RNU44 and U6. Quantification of miR15 and miR16 was done using a TaqMan assay (Applied Biosystems). Changes in expression were calculated using the ΔΔCT method. Cells t ransfected with mir-CTR were used as calibrator.
Apoptosis assays
Cultured cells were double stained with Annexin V-FITC conjugate (BD Biosciences Pharmingen, San Josè, CA, USA), and PI in isotonic solution, and then analyzed by FC. Viable cells were defined as double negative cells(53).
Xenogeneic mouse transplantation
A xenograft model for CLL growth was used for in vivo experiments (54, 55). All procedures involving animals were performed in the respect of the current National and International regulations and were reviewed and approved by the Licensing and Animal Welfare Body of the IRCCS-AOU San Martino-IST National Cancer Research Institute, Genoa, Italy.
In miRNA pre-treatment experiments 2-3 NSG mice (according the number of cells available) were inoculated with CLL cells transfected with miRNA mimic/inhibitors and cultured for 6 h prior to injection. A total of 50x106 CLL cells per mouse were injected together with a proportion of autologous T cells (approximately 5-10%).
After four weeks, mice were anesthetized by intraperitoneal injection of combination of xylazine (10 mg/kg) and ketamine (100 mg/kg) and analyzed by Magnetic Resonance Imaging (MRI) with USPIO contrast reagent(56).
We decided to end the experiments maximum 6 weeks from start. The Animal Welfare Body posed a time limit to the experimental protocol to prevent unneeded suffering.Animals were sacrificed in a saturated CO2 chamber and autopsies were performed. Spleens were evaluated by FC and by immunohistochemical (IHC) analysis.
Single cell suspension form spleens were obtained by using gentleMACS™ Dissociator (Miltenyi). Cells were stained with anti-human CD45-FITC, CD19-PECy7, CD5-APC, (BD Biosciences) and analyzed by FC. Apoptosis was evaluated using Annexin-V-FITC, CD19-PE-Cy7, CD5-PE, CD45-APC cell staining (BD Biosciences).
Part of the spleens were not used for dissection but they were formalin-fixed. Paraffin-embedded spleen samples were then analyzed for the presence of human cells ( both CLL cells and T lymphocytes) (56) by using anti-CD20 Mouse monoclonal antibody
(clone L26) and CD3 Rabbit Monoclonal Antibody (clone 2GV6). Ultraview Universal Red Detection Kit (Ventana Medical System) was used. The sections were evaluated by two observers with an Olympus light microscope using 4X, 10X, 40X and objectives under a Leica DMD108 optical digital microscope (Leica Microsystems).
In the experiments in which we want to evaluate therapeutic effects of miRNA, CLL engraftment in the spleen was determined by MRI following USPIO contrast reagent injection after 2-3 weeks from cell injection(56). At this stage, mice were treated intraperitoneally [every second day with mirVana™ miRNA mimic, (In Vivo Ready formulation, Ambion Inc)] complexed with Invivofectamine 2.0 (Thermo Fisher Scientific) at a final concentration of 0.7 mg/mL (200 µL/mouse). Overall three doses were administered. The following miRNA were used: hsa-miR15a-5p, hsa-miR16-5p, miRNA negative control#1. Three days from the last injection, mice were analyzed again by MRI and then sacrificed in a saturated CO2 chamber and autopsies were performed. Spleens were analyzed by FC and IHC. Apoptosis was evaluated by Annexin-V-FITC, CD19-PE-Cy7, CD5-PE, CD45-APC, and FC or by Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and IHC on spleen tissue sections.
Magnetic Resonance Imaging
All in-vivo MRI experiments and MRI analyses, using USPIO nanoparticles, were carried out and acquired as previously described(56).
Ig /TCR clonality testing
Splenic cells suspension of mice inoculated with CLL cells were investigated after autopsy for the presence of leukemic cells by using a molecular PCR approaches. BcR was amplified with primers specific for IGHV Fr1 region or Fr2 or Leader region in conjunction
with primers IGHJ (or primers specific for the constant mu and gamma chain in case of RNA analysis). For TcR rearrangements primers of BIOMED were used.(57, 58)
Definition of IHC index
The IHC index is a measure of the spread of leukemia based on the average diameters of the follicular lesions. An arbitrary value of 1 to was assigned to the follicles with diameters±SD of 102(±90) x 42(±7) µm; a value of 3 to follicles with a diameter 195(±80) x 138(±85) µm; a value of 6 to follicles between 399(±245) x 300(±39), and a value of 12 to follicles between 734(±461) x 540(±167). The IHC index is given by the sum of the number of follicles multiplied by the value assigned according to size (see results).
Statistical analysis
SPSS for Windows, v13.0, 2004 software (SPSS UK) was used for all analyses. For categorical variables, statistical comparisons were performed using two-way tables for the Fisher’s exact test and multiway tables for the Pearson’s Chi-square test. Statistical comparisons between related samples were carried out by Wilcoxon test or by Mann-Whitney U for unpaired samples. A value of p<0.05 was considered significant for all statistical calculations.
RESULTS
Comparison of miRNA expression between del13q CLL and others CLL
Previous analysis of miRNAome array demonstrated that in the CLL many microRNA are deregulated in comparison to normal B cell subsets(36). Among these miRNAs, miR15 and miR16, were very interesting for their implication in the pathogenesis. Supervised analysis of the expression of mir15 and miR16 in 203 CLL patients at Binet A stage was performed. At disease onset, only miR16 reached a statistical significance (p<0.0001) in del13q patients versus others CLL (Figure 1a). Patients with del13q were further subdivided into different groups basing of the heterogeneity of the deletions (i.e. mono allelic deletion ; biallelic deletion ; a mixture of mono/biallelic. Figure 1b shows a FISH analysis of the above subsets). A significative lower level of both miRNAs was found in bi-allelic CLL compared to mono bi-allelic CLLs (Figure 1c). Data were confirmed by qRT-PCR (Figure 1d ).
Transfection of miR15 and miR16 mimics in CLL cells in vitro
performed by electroporation and checked by flow cytometry using smartflare technology and qRT-PCR. SmartFlare™ probes are designed to fluoresce when they detect specific targets in live cells (i.e. PI- negative cells). By using RNA as the target, SmartFlare™ Probes allow to identify and analyze various markers in live cells with a single incubation step. After the incubation, the probes exit the cells allowing to perform downstream analyses with the same, unperturbed cells (Figure 2a). In the control preparations, CLL cells were transfected with random nucleic acid sequences with no identifiable miRNA function (miR-CTR or miR-CTR inhibitor). Figure 2b shows a typical staining performed on a CLL sample after 6 hrs of incubation with Smartflare probe. The methodology also allows to evidence the increase of miRNA expression after transfection. (Figure 2a middle and bottom line)
We transfected 7 CLL cells with biallelic del13q by electroporation as detailed in metohs. Both miR15 and miR16 were significantly higher than control preparations. (p= 0.015; Figure 2c). The entry of miRNA also was evaluated by qRT–PCR (Figure 2d). Transfection of miR15 and miR16 inhibitors in CLL cells in vitro
For the experiments with miRNA inhibitors, we selected 6 CLL cases (characterized by a variable extent of Mir15 and Mir16 expression evaluated by qRT-PCR(not shown) from the O-CLL protocol that did not display biallelic del13q. Transfection with specific miRNA inhibitors reduced miRNAs expression in all CLL cells tested compared to control preparations (p=0.03; Wilcoxon test) (Figure 3a). Figure 3b shows an example of this inhibition. The value of inhibition was validated by qRT-PCR at 24 hrs after transfection(not shown).
Transfection of miR15 and miR16 interferes with CLL survival in vitro
Since miR15 and miR16 expression may impact on CLL cell survival in vitro, purified cells from 12 CLL cases with biallelic del13q were transfected with miR15 or
miR16 miRNA mimics and cultured up to 72 hours. Viable cells, defined as cells negative for Annexin-V (apoptotic cells) or PI (necrotic cells), were measured at different time intervals. Figure 4a reports a typical time course experiment. Transfection of miR15 or miR16 caused a significant decrease of cell viability (53.32±24 for miR15; p=0.001 and 47.74± 21 for miR16 mimic p=0.0015, respectively) in 11/12 CLL samples at 48 hours time point (Figure 4b). Figure 5a report data of single cases used for miRNA transfection. Viability of cells from GC0620 CLL case did not change following successful transfection of miR15 or -16. Interestingly, this CLL case displayed a p53 mutation(Exon 6; C.626_627DELGA). Two other CLL cases (MG0248, PA0254), displaying p53 mutations, albeit of a type different from that observed in GC0620 CLL cells ( EXON 8; C.844C>T and EXON 5 C.481G>A, respectively), had a substantial drop in cell viability following transfection with either miR15 or miR16. All these cases, harboring TP53 mutations are in green area in Figure 5a.
Transfection of miR15 or miR16 into del13q CLL cells resulted consistently into down modulation of proteins such as BCL2 or MCL1 (anti-apoptotic) and Cyclin D1 or D2 (cell cycle induction) encoded by genes which represent targets of these miRNAs cluster (37, 38). Survivin, which is involved in a different apoptotic pathway, was not down regulated (Figure 5b ).
In contrast, inhibition of miR15 or miR16 expression, resulted into a substantial increase of CLL cell viability. CLL cases(not shown). The viability increase was observed in all the cases irrespective to their chromosomal abnormalities showing that miRNA inhibition is always related to a better survival for the cells.
For in vivo analysis we searched different methodologies capable to give some indication of disease engraftment before the treatment. We decided to use a methodology based on iron oxide-magnetic resonance imaging (USPIO-MRI). Iron uptake is inversely correlated to the presence of CLL follicles-like structures, which prevent iron uptake. Signal to noise ratio (SNR%) is higher when low iron enter into the spleens. Figure 6 shows an example of MRI imaging and their relation with engraftment of the disease. Figure 6b-c highlights the absence of CD20+ cells in NSG-CTR mouse and the presence of focal aggregates of CD20 positive cells in NSG-CLL spleen. In addition, Perls’ Prussian blue staining (used to detect USPIO nanoparticles) indicated that ferric iron particles were excluded from the focal lesions (Figure 6e) whereas a random distribution of USPIO nanoparticles was observed in the spleens of NSG-CTR mice. USPIO-MR was usually used after 2 and 4 weeks the CLL cells injection as described in dept in (56). All the mice included in the in vivo experiments also were analyzed by IC. Typically, multiple foci of CD20+ B cells surrounded by a ring of T cells were observed. T cells were mostly CD4+ (Figure 7). To confirm the presence of leukemic clone in the lesions, all samples were analyzed for BcR and TCR repertoire (Figure 7b). The presence of a unique BcR pick indicated the presence of the leukemic clone in the spleen mice while TcR showed a profile similar between CLL patients and CLL cells isolated from mice spleen. Since we needed a measure to quantify the engraftment of the disease, we designed an arbitrary immunohistochemical index or IHC index as reported( Figure 8).
Injection of CLL pre-treated with miRNA-15 or 16 mimics or inhibitors in vitro influences the growth of CLL cells in NSG mice
CLL cells with biallelic del (13)(q14) (cases CD0310, GD0051, RD90296, MG0248) were purified and transfected with miR15, miR16 or miR-CTR mimics, cultured for 6 hours and injected i.v. into NSG mice together with autologous T cells (10:1 B cells/Tcell ratio) 2- 3 mice were used in each treatment group . The spleens of NSG mice receiving
miR15 or miR16 mimics pre-treated CLL cells displayed a higher uptake of USPIO (mean ± s.d. = − 31.1 ± 25.1 andmean ± s.d.= − 18.2 ± 28.19 for miR15 and miR16, respectively) contrast reagent , corresponding to a lower SNR %, compared to the spleens of control mice receiving miR-CTR-treated CLL cells (+30.7 ± 23.4) (Figure 9a). The SNR % of miR15/16-treated mice resembled that of NSG mice that had not received leukemic cells(-54.3 ±15). This MRI pattern was related to the lower number of neoplastic foci within the splenic white pulp as also measured by immunohistochemistry with anti-CD20 Ab. In fact, the spleens of mice injected with miRNA mimics-preatreated CLL cells exhibited significantly lower proportions of CD19+CD5+ CLL cells compared to controls (p=0.012 ). In these mice, the IHC index was decreased compared to that of NSG mice receiving control cells (means not shown; example of IHC index is reported in Figure 9b). The spleens of mice injected with miRNA mimics-preatreated CLL cells exhibited significantly lower proportions of CD19+CD5+ CLL cells compared to controls (p=0.012 ). (Figure 9c) while no decrease of T cells was observed. B cells recovered from NSG mice consistently shared identical BcR gene rearrangement with the leukemic clone whereas T cells were oligoclonal (data not shown).
In another set of experiments, the cells from two CLL cases (PM0608, PA0145), that were monoallelic for del13q, were pre-treated with miR15 or miR16 inhibitors in vitro and subsequently injected into NSG mice together with T cells (see above). With this treatment we observed a large infiltration of the spleen by neoplastic foci. The average of IHC index was increased of 69±6 and 71±17 respectively for miR15 or miR16 inhibitor pre-treated cells. (Figure 10b). Flow cytometry showed an increase of CD45+CD19+CD5+ cells of 71±3% and 69±1% with pre-treatment of miR15 or miR16 inhibitors respectively (Figure 10c). Again, BCR and TCR gene rearrangement analysis confirmed that the engrafted B cells were mostly from the leukemic clones, whereas T cells were oligoclonal (not shown).
Inhibition of CLL growth after miR15 and miR16 mimics administration
The capacity of miR15 and 16 to control CLL growth in vitro and in vivo suggests a possible usage of these molecules for CLL therapy. In order to evaluate whether miRNA mimics were able to enter inside splenic CLL cells and to inhibit tumor growth, a pilot experiment was designed. MG0248 CLL cells were inoculated into six NSG mice and after four weeks, a single dose of miR15 , miR16 or miR-CTR complexed with Invivofectamine was administered intraperitoneally (i.p.). Mice were sacrificed 24-h after miRNA administration; splenic cell suspension were obtained and cells were stained for CD45+, CD19+ and CD5+ and purified by FACS sorting. The entry of miRNAs was evaluated by qRT-PCR that showed an high increase of miR15 (fold increase 198 and 841 respectively) and of miR16 (fold increase 119 and 5,6) levels in the two treated mice versus control mice (mice injected with miR-CTR(Figure 11).
Next, CLL cells from six cases with biallelic del(13)(q14) were inoculated into NSG mice together with T cells (NSG-CLL) and CLL engraftment was verified in the spleen after approximately two weeks by MRI(56). MRI was used for determination of mice to place on treatment by selecting only mice with ΔSNR% above to NSG mice that had not received leukemic cells (NSG-CTR, n=12, ΔSNR% −54.3±15). Treatment consisted of a total of three injections on alternate days. Three days after the last treatment, splenic infiltration by leukemic cells was evaluated by MRI : miR-CTR treated mice displayed a splenic infiltration by leukemic cells with an 63±14 average increase of ΔSNR% over that of mice not receiving leukemic cells (p <0.0001). Briefly, mice treated with miR15 or miR16 mimics showed a significant ΔSNR% decrease (p<0.0001 and p=0.0002, respectively) compared to mice before therapy administration while miR-CTR showed an 63±14 average increase of ΔSNR% over that of mice not receiving leukemic cells (p <0.0001) (Table 1). IHC
analysis showed an almost complete disappearance of the typical aggregates of leukemic (CD20+) cells after treatment with miR15 or miR16 mimics (Figure 12a). However, autologous T cells (CD3+ cells), surrounding what was presumably the area of the previously existing CLL cell aggregates/nodules, were still present possibly indicating that T cells were not affected by treatment (Figure 12b). A significantly lower IHC index was observed in mice treated with miR15 (62±15 (p=0.0007)) or miR16 78±22 (p=0.02) compared to mice treated with miR-CTR (Table 1). These results were concordant with FC analysis that showed a reduction of CLL cells after miR15 and miR16 mimics treatment, compared with miR-CTR (average % of 61±8 (p=0.0006) and of 75±12 (p=0.0001)) as reported in Figure 12c.
The presence of "empty nodules" suggested apoptotic leukemic cells. This hypothesis was supported by the presence of cleaved CASP3 in the “empty nodules” (not shown) and by the observation of an increased percentage of Annexin V positive CLL cells (p<0.0001) in the splenic cell suspension by FC (Figure 12d).
Finally, same experiments were performed in CLL that did not have biallelic deletion. Briefly, NGS mice were inoculated into 5 CLL patients : 2 were monallelic for del13q CLL, one case with Trisomy 12 and the last with both del17p- and somatic mutation of TP53. Following miR15 or miR16 mimic treatment, a significant reduction of CD45+CD19+CD5+ cells was observed by FC (p=0.0001 and p=0.006, respectively compared to miR-CTR) (Table 2). Again a decrease in the IHC index of 72±18% and 74±10% following treatment with miR15 and miR16 mimics, respectively (p=0.001) was observed in all cases except the patient with dysfunctional P53.
DISCUSSION
This study demonstrates that transfection of miR15 and miR16 mimics is capable to decrease the cell viability of pre-treated CLL both in vitro and in vivo. Moreover, miR15 and miR16 mimics cause tumor regression in NGS mice previously engrafted with CLL cells. Both observations support previous data of the capacity of miR15 and miR16 to control apoptotic pathway and proliferative property of CLL cells. (35, 37, 38, 45).
In order to evaluate the central role of miR15 and 16 in controlling clonal expansion in all CLL cells, the experiments were not limited to patients lacking of miR15 and miR16 for genetic anomalies (biallelic del13q), but were extended to different CLL patients in which the presence of endogeneous miR15 and miR16 was potentially allowed (i.e. normal FISH, monoallelic del13q, other lesion). Once again, the inhibition of miR15 and 16 increased cell survival in vitro and caused a significant tumor expansion in vivo. In contrast, by enforcing miR15 and 16 expression, an impaired survival in vitro and diminished expansion of tumor in NGS mice was observed. These results indicate the central role played by miR15 and miR16 in clonal expansion of full-blown CLL. Notably, this capacity seems to be sustained by the only miR15 and miR16, without involvement of DLE2 or DLEU7 (that are located on del13q), thus reinforcing the notion that these miRNAs play a crucial role in CLL development per se’.
In order to exclude that exogenous miRNA transfection could interact with B-T cell signals, that are crucial for survival of CLL cells, in a series of experiments, B cells were first separated from T cells, then transfected them with miRNAs. B and T cells were finally put together before in vivo injection. In fact, CLL growth in NGS mouse strongly depend from a correct ratio of T/B cells ratio(55).
CLL B cells, before injection into mice. We could not observed an unaltered distribution pattern of T cells while an important diminished proportion of B cells was noticed.
For the experiments in which miR15 or miR16 mimics were used in vivo, after engraftment of the disease, we noticed an unaltered T cell proportions surrounding different “empty” areas, previously occupied by CLL cells (Figure 12b). This observation support the idea that alteration of CLL cells growth is related to their apoptotic/survival apparatus instead to signal receiving from interaction with T cells.
Finally, it should be discuss the role of miRNAs as simple biomarkers for detecting new target therapy or their potential usage in therapy (18, 36, 44, 59).
The encouraging findings are that CLL cells engrafted in mice can easily adsorb the
miRNAs administered in a liposomal form and that the miRNA mimics are effective also on cells with additional chromosomal alterations. Clearly, the problem of whether these
reagents are effective on a putative leukemic stem cells may be of relevance and does not have a solution at present. An additional problem may be represented by the TP53 gene status and functioning in the leukemic cells. Indeed, in this study, some of the results of the in vivo and in vitro experiments showed that the presence of TP53 gene
mutations/deletions may render the miR15 and miR16 mimics administration less effective. This was not unexpected given the close interactions occurring between miR15 and
miR16, TP53 and the cluster of miR-34 genes in the regulation of cell
survival/apoptosis(60) . However, there are not so far sufficient CLL samples tested to delineate clearly as TP53 lesions are capable of rendering the usage of miR15 and miR16 ineffective. More experiments along this line are currently in progress. These observations are being extended to additional cohorts of patients including more advanced
stage/relapsed patients that are comprised of more numerous cases with TP53 alterations. Thus, the miRNA approach, especially if multiple miRNA mimics and inhibitors can be
targeted, either alone or in combination with other drugs, may represent an additional therapeutical strategy
REFERENCES
1. Dighiero G & Hamblin TJ (2008) Chronic lymphocytic leukaemia. Lancet 371(9617):1017-1029.
2. Chiorazzi N, Rai KR, & Ferrarini M (2005) Chronic lymphocytic leukemia. N Engl J Med 352(8):804-815.
3. Rozman C & Montserrat E (1995) Chronic lymphocytic leukemia. N Engl J Med 333(16):1052-1057.
4. Osorio LM, Jondal M, & Aguilar-Santelises M (1998) Regulation of B-CLL apoptosis through membrane receptors and Bcl-2 family proteins. Leuk Lymphoma 30(3-4):247-256.
5. Stilgenbauer S, Lichter P, & Dohner H (2000) Genetic features of B-cell chronic lymphocytic leukemia. Reviews in clinical and experimental hematology 4(1):48-72. 6. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, & Stevenson FK (1999) Unmutated Ig V(H)
genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94(6):1848-1854.
7. Damle RN, et al. (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94(6):1840-1847.
8. Rassenti LZ, et al. (2008) Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia. Blood 112(5):1923-1930.
10. Agathangelidis A, et al. (2012) Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood 119(19):4467-4475.
11. Baliakas P, et al. (2014) Clinical effect of stereotyped B-cell receptor immunoglobulins in chronic lymphocytic leukaemia: a retrospective multicentre study. Lancet Haematol 1(2):e74-84.
12. Stamatopoulos K, Agathangelidis A, Rosenquist R, & Ghia P (2017) Antigen receptor stereotypy in chronic lymphocytic leukemia. Leukemia 31(2):282-291.
13. Duhren-von Minden M, et al. (2012) Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature 489(7415):309-312.
14. Minici C, et al. (2017) Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia. Nat Commun 8:15746.
15. Dohner H, et al. (2000) Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 343(26):1910-1916.
16. Kalachikov S, et al. (1997) Cloning and gene mapping of the chromosome 13q14 region deleted in chronic lymphocytic leukemia. Genomics 42(3):369-377.
17. Rawstron AC, et al. (2008) Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N Engl J Med 359(6):575-583.
18. Morabito F, et al. (2013) Clinical monoclonal B lymphocytosis versus Rai 0 chronic lymphocytic leukemia: A comparison of cellular, cytogenetic, molecular, and clinical features. Clin Cancer Res 19(21):5890-5900.
19. Dohner H, et al. (1997) 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis. Blood 89(7):2516-2522.
20. Neilson JR, et al. (1997) Deletions at 11q identify a subset of patients with typical CLL who show consistent disease progression and reduced survival. Leukemia 11(11):1929-1932.
21. Grever MR, et al. (2007) Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US Intergroup Phase III Trial E2997. J Clin Oncol 25(7):799-804.
22. Stilgenbauer S, et al. (2009) Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol 27(24):3994-4001.
23. Fischer K, et al. (2012) Bendamustine in combination with rituximab for previously untreated patients with chronic lymphocytic leukemia: a multicenter phase II trial of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol 30(26):3209-3216.
24. Zenz T, et al. (2009) Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood 114(13):2589-2597.
25. Moreno C & Montserrat E (2010) Genetic lesions in chronic lymphocytic leukemia: what's ready for prime time use? Haematologica 95(1):12-15.
26. Chiaretti S, et al. (2014) NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukemia undergoing first-line treatment: correlation with biological parameters and response to treatment. Leuk Lymphoma 55(12):2785-2792. 27. Lagos-Quintana M, Rauhut R, Lendeckel W, & Tuschl T (2001) Identification of novel
28. Ghildiyal M & Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10(2):94-108.
29. Hausser J & Zavolan M (2014) Identification and consequences of miRNA-target interactions--beyond repression of gene expression. Nat Rev Genet 15(9):599-612. 30. Ambros V (2003) MicroRNA pathways in flies and worms: growth, death, fat, stress,
and timing. Cell 113(6):673-676.
31. Calin GA, et al. (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A 101(32):11755-11760.
32. Calin GA, et al. (2002) Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99(24):15524-15529.
33. Ouillette P, et al. (2008) Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14. Cancer Res 68(4):1012-1021.
34. Mosca L, et al. (2010) Integrative genomics analyses reveal molecularly distinct subgroups of B-cell chronic lymphocytic leukemia patients with 13q14 deletion. Clin Cancer Res 16(23):5641-5653.
35. Sellmann L, et al. (2010) Gene dosage effects in chronic lymphocytic leukemia. Cancer Genet Cytogenet 203(2):149-160.
36. Negrini M, et al. (2014) microRNAome expression in chronic lymphocytic leukemia: comparison with normal B-cell subsets and correlations with prognostic and clinical parameters. Clin Cancer Res 20(15):4141-4153.
37. Calin GA, et al. (2008) MiR-15a and miR-16-1 cluster functions in human leukemia. Proc Natl Acad Sci U S A 105(13):5166-5171.
38. Cimmino A, et al. (2005) miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A 102(39):13944-13949.
39. Linsley PS, et al. (2007) Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression. Mol Cell Biol 27(6):2240-2252.
40. Allegra D, et al. (2014) Defective DROSHA processing contributes to downregulation of MiR-15/-16 in chronic lymphocytic leukemia. Leukemia 28(1):98-107.
41. Veronese A, et al. (2015) Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia. Leukemia 29(1):86-95.
42. Mertens D, et al. (2006) Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-suppressor mechanism. Proc Natl Acad Sci U S A 103(20):7741-7746.
43. Raveche ES, et al. (2007) Abnormal microRNA-16 locus with synteny to human 13q14 linked to CLL in NZB mice. Blood 109(12):5079-5086.
44. Calin GA, et al. (2005) A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 353(17):1793-1801.
45. Klein U, et al. (2010) The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell 17(1):28-40.
46. Underbayev C, et al. (2016) Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia. Oncotarget.
47. Pekarsky Y & Croce CM (2015) Role of miR-15/16 in CLL. Cell Death Differ 22(1):6-11. 48. Mott JL, Kobayashi S, Bronk SF, & Gores GJ (2007) mir-29 regulates Mcl-1 protein
expression and apoptosis. Oncogene 26(42):6133-6140.
49. Pekarsky Y, et al. (2006) Tcl1 expression in chronic lymphocytic leukemia is regulated by miR-29 and miR-181. Cancer Res 66(24):11590-11593.
50. Morabito F, et al. (2015) Is ZAP70 still a key prognostic factor in early stage chronic lymphocytic leukaemia? Results of the analysis from a prospective multicentre observational study. Br J Haematol 168(3):455-459.
51. Lionetti M, et al. (2014) High-throughput sequencing for the identification of NOTCH1 mutations in early stage chronic lymphocytic leukaemia: biological and clinical implications. Br J Haematol 165(5):629-639.
52. Cutrona G, et al. (2017) Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy. Leukemia.
53. Cutrona G, et al. (2008) Clonal heterogeneity in chronic lymphocytic leukemia cells: superior response to surface IgM cross-linking in CD38, ZAP-70-positive cells. Haematologica 93(3):413-422.
54. Durig J, et al. (2007) A novel nonobese diabetic/severe combined immunodeficient xenograft model for chronic lymphocytic leukemia reflects important clinical characteristics of the disease. Cancer Res 67(18):8653-8661.
55. Bagnara D, et al. (2011) A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease. Blood 117(20):5463-5472.
56. Valdora F, et al. (2016) A non-invasive approach to monitor chronic lymphocytic leukemia engraftment in a xenograft mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI). Clin Immunol.
57. Colombo M, et al. (2011) Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia. Mol Med 17(7-8):834-839.
58. Langerak AW, et al. (2012) EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia 26(10):2159-2171.
59. Van Roosbroeck K & Calin GA (2016) MicroRNAs in chronic lymphocytic leukemia: miRacle or miRage for prognosis and targeted therapies? Semin Oncol 43(2):209-214.
60. Fabbri M, et al. (2011) Association of a microRNA/TP53 feedback circuitry with pathogenesis and outcome of B-cell chronic lymphocytic leukemia. JAMA 305(1):59-67.
