ALBERT

Description: B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are postulated to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 28 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 96% (27/28) of patients. The most frequent rearrangements were observed in IGH genes (96%), including complete IGHV-IGHJ in 75% (21/28) and incomplete IGHD-IGHJ in 31% (8/28) of patients. Among complete IGH rearrangements 4 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 20 patients (71%) (including 25% of IGKV-IGKJ, 50% of IGKV/intron-Kde, and 25% of IGLV-IGLJ) indicating active receptor editing occurring during B lymphoblasts leukemogenesis. Cross-lineage TCR genes rearrangements were found in 77% (23/28) of patients. TCR beta genes rearrangements were detected in 46% (13/28) of patients (complete TRBV-TRBJ in 32% (9/28), TRBD-TRBJ in 5/28 patients - 18%). TRGV-TRGV were found in 46% (13/28), TRDV-TRDJ in 50% (14/28; 10 monoallelic and 4 biallelic). TCR beta genes rearrangements with presence of TCR gamma genes rearrangements were identified in 25% (7/28) of patients. The identified Ig and TCR rearrangements were stable in patients monitored for minimal residual disease (MRD) and patients with leukemia relapse. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblasts undergo many rearrangements on the same IGK allele before they rearrange IGL genes. The data suggest the role of antigen in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as a possible molecular marker for monitoring MRD in B-ALL.

Description: Allogeneic hematopoietic stem cell transplantation (alloHSCT) in chronic phase of chronic myeloid leukemia (CML) is associated with long-term disease-free survival and potentially eradication of leukemic cells. The goal of early MRD detection is to allow timely therapeutic intervention before hematologic relapse. The concomitant detection of BCR-ABL mRNA following alloHSCT is strongly associated with relapse, though not absolutely predictive. The relapse risk decreases with increased time after alloHSCT. The detection of BCR-ABL mRNA is the most strongly associated with relapse shortly after HSCT but all patients need to be monitored indefinitely after transplantation by molecular techniques presumably at 3–6 months intervals. Real-time quantitative PCR (RQ-PCR) for BCR-ABL mRNA provides an accurate and reliable measure of response to therapy in CML. In this study we evaluated 412 available samples from 75 patients at 1 month to 10 years after allo HSCT. Quantification of BCR-ABL was performed by RQ-PCR assay according to the Europe Against Cancer (EAC) protocol. Peripheral blood/bone marrow samples were studied every 3–6 months after alloHSCT for the presence of BCR-ABL transcripts using RT-PCR/nested PCR and RQ-PCR. RT-PCR positive patients were analyzed further at monthly intervals. RNA isolation from mononuclear cells was performed by column method. Reverse transcription was performed using Super Script II and random hexamers. BCR-ABL level was normalized with control ABL gene and expressed as the ratio of BCR-ABL/ABL compared to diagnostic sample or median expression values of BCR-ABL/ABL from EAC protocol. In our group BCR-ABL/ABL ratio decreased at least 1000-fold in all patients after alloHSCT. RT-PCR became negative in 64.7% patients after first 90 days. In the group of 65 patients with RQ-PCR tests performed at least 1 year after alloHSCT, 12 (18.5%) patients were always negative (no BCR-ABL/ABL transcripts detected, at least 10−5 test sensitivity), 40 (61.5%) were persistently low-level positive (with the BCR-ABL/ABL ratio less than 0.02%) and 13 (20.0%) patients were stable, high-level positive with transcript levels exceeded 0.02% threshold. The molecular relapse was observed in three patients with 15–80 fold increased of BCR-ABL expression in the first year after SCT. Decreased immunosuppressive therapy allowed achieving molecular remission in two patients. One patient developed hematological relapse despite donor lymphocyte infusion (DLI). One patient developed cytogenetic relapse and was successfully treated with 400 mg imatinib daily dose and achieved molecular remission with a low-level BCR-ABL expression. Standard imatinib therapy was applied in seven patients prior to SCT without negative transplant outcome. We conclude that RQ-PCR is valuable method to quantitate BCR-ABL expression in CML patients after alloHSCT and allows monitoring the kinetics of BCR-ABL mRNA transcipts and is useful in prediction of the hematologic relapse.

Description: Four women aged from 48 to 51 years with persistent polyclonal B-cell lymphocytosis (PPBCL) were studied for the clonality of B-cell population and HLA haplotype. All patients were smokers, one presented an immune deficiency syndrome due to decreased IgG and IgA levels, the others were asymptomatic. All patients presented serum increase of polyclonal IgM (5.55 – 8.12 g/L), absolute peripheral lymphocytosis ranging from 6100 to 10500/μL with elevated proportion of B cells, the percentage of CD5/CD19 lymphocytes 〈 5%, and binucleated/bilobulated lymphocytes accounting for 5–9% of nucleated cells on the blood smear. In one patient, cytogenetic study revealed trisomy 3 in 4 out of 40 metaphases, +18 in 2 cells, and i(18q), +15, t(13,14) in single cells. In the remaining patients no clonal chromosomal abnormalities were found. All patients were HLA typed by PCR based sequence specific primer amplification. HLA allele and haplotype incidence in patients’ group were compared to healthy population of 286 ethnically matched (Caucasian, Polish) controls. DRB1*07 allele was found in 2/4 patients, seems than to be elevated than in general Polish population but a small number of patients does not allow to perform relevant statistical tests. Moreover we found significantly more frequent B*08-DRB1*03 fragment of ancestral HLA 8.1 haplotype (8.1 AH) in PPBCL patients (3/4) than in control group (47/286) (OR=15.3; 95% CI 1.55–150; p=0.02). Peripheral blood mononuclear cells from all PPBCL patients were also studied for monoclonality in immunoglobulin (IGH VH-JH, incomplete DH-JH, IGK, IGL) and crosslineage TCR (TCRB, TCRG, TCRD) genes rearrangements according to BIOMED-2 protocol. The multiplex PCR reactions were followed with heteroduplex analysis of PCR products. We found polyclonality of lymphocytes population in all tests except incomplete DH7-JH IGH rearrangements in 2 patients. The described gene rearrangement was not related to other upstream DH or downstream JH gene segments. The HLA incidence we found is distinct from previously described elevated incidence HLA DRB1*07 allele and may suggest another genetic background of PPBCL pathogenesis. Moreover, our finding of high prevalence of 8.1 AH, known to be more frequent in autoimmune diseases, and to worsen the prognosis in some infectious diseases and non-Hodgkin lymphomas, as well as of the incomplete DJ monoclonality in 2/4 patients, warrant further studies aiming to determine the relationship between PPBCL, immune disorders and true lymphoproliferative diseases.

Description: The immunoglobulin gene rearrangements occur during early lymphoid differentiation in hierarchical order. First the immunoglobulin heavy chain genes (IGH) rearrange, then IGK and in case of IGK deletion rearrange IGL genes. All B-lymphocytes with IGL rearranged genes should have monoallelic or biallelic IGK genes deletions. The human IGK locus contains several variable genes (IGKV), joining genes (IGKJ), genes coding constant region (IGKC), and DNA fragments involved in recombination described as kappa deleting element (Kde). Both IGKV genes and recombination signal sequences (RSS) in the JK-CK region can rearrange to Kde creating respectively IGKV-Kde or intron RSS-Kde rearrangements. Kde rearrangements inactivate IGK allele causing the deletion of either the CK (intron RSS-Kde) or the entire JK-CK region. IGK and IGL rearrangements were analyzed in 48 patients with B-lymphocyte chronic lymphocytic leukemia (B-CLL) from peripheral blood/bone marrow mononuclear cells or enlarged diagnostic lymph nodes. Rearrangements of IGK and IGL were detected in multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol (IGKV-IGKJ, IGKV/intron-Kde, and IGLV-IGLJ). In total, 41 IGK and IGL rearrangements were identified in 46 patients (89.1%). In 5 patients (10.9%) rearrangements were not found - including 4 DNA samples extracted from paraffin embedded tissue and one DNA sample isolated from peripheral blood. IGK rearrangements were detected in 37 of 46 patients (80.4%). In 24 patients (52.2%) IGK rearrangements without presence of IGL rearranged genes were identified, including 18 (39.1%) with only IGKV-IGKJ and 6 (13.1%) with IGKV-IGKJ and also IGKV/intron-Kde. Parallel rearranged IGK and IGL genes were found in 13 (28.3%) patients: 8 (17.4%) patients with IGKV-IGKJ and IGKV/intron - Kde and IGLV-IGLJ, 1 (2.2%) patients with IGKV-IGKJ and IGLV-IGLJ, 4 (8.7%) patients IGKV/intron-Kde and IGLV-IGLJ. In IGKV/intron-Kde rearranged genes group were detected the following rearrangements: IGKV1f/6/IGKV1-Kde (4 of 17); IGKV3f/intron Kde (13 of 17); IGKV2f/IGKV4/IGKV5-Kde (3 of 17; 17 monoallelic and 3 biallelic forms). Rearrangements with Kde always coexisted with IGKV-IGKJ or IGLV-IGLJ. In 4 (8.7%) patients IGL rearrangements were found without any IGK rearranged genes - three (6.5%) DNA samples were obtained from paraffin embedded tissue and the last DNA sample was isolated from peripheral blood after therapy. Lack of IGK monoclonality detection in paraffin embedded tissue samples resulted probably from low DNA quality. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL rearrangements incidence and present allelic exclusion and active receptor editing in B-CLL patients. The data provide additional evidence for the role of antigen in B-CLL immunopathogenesis. B-CLL lymphocytes undergo many rearrangements on the same IGK allele before they rearrange IGL genes and produce lambda chains. The results indicate also rearranged IGK/IGL genes as a possible molecular marker for monitoring minimal residual disease in B-CLL.

Description: Introduction The human leucocyte antigen-G (HLA-G) is a nonclassical class Ib molecule that suppresses various immune cell functions and may contribute to immune escape and cancer development. HLA-G polymorphisms, especially HLA-G-725(C/G/T) 5’URR and HLA-G 14bp del/ins 3’UTR, might influence the expression of HLA-G transcript and protein, and in consequence, affect the biological features of HLA-G. Therefore, we investigated whether these two polymorphisms, which seem to be functionally relevant, may play a role in susceptibility to chronic lymphocytic leukemia (CLL) and a clinical course of the disease. So far, no studies have reported any potentially impact of HLA-G polymorphisms on lymphoid neoplasms. Methods HLA-G725(C/G/T) 5’URR and HLA-G 14bp del/ins 3’UTR polymorphisms were genotyped in 167 previously untreated patients with CLL. The control group consisted of 98 randomly selected blood donors. Results Strong linkage disequilibrium between HLA-G-725(C/G/T) and HLA-G 14 bp del/ins was observed (D’=1.0 and r2=0.2). Six distinct haplotypes, including G/del, C/del, T/del, G/ins, C/ins, T/ins were found in the CLL patients. Among the controls only five haplotypes were found due to the T/ins haplotype not being observed. The probability of the occurrence of G/ins and T/ins haplotypes was higher in the CLL than in the controls (p= 0.01). The analysis of the prognostic significance of diplotypes, as well as the previously reported correlations between HLA-G genotypes and HLA-G expression in vitro and in vivo, allowed us to identify the HLA-G diplotype-based risk groups. The low-risk (LR) group comprised CC/del-del, CC/del-ins and CC/ins-ins diplotypes, and the high risk (HR) group included GG/del-del, GG/del-ins, GG/ins-ins, GC/del-del, GC/del-ins, GC/ins-ins, TT/del-del, TT/del-ins, TT/ins-ins and CT/del-ins diplotypes. The patients carrying LR diplotypes presented a higher 3-year treatment-free survival (TFS) (56.7%, 95% CI 47-66) than those with HR diplotypes (38.6%, 95% CI 27-52; p= 0.005). Additionally in the group of mutated IGHV patients, subjects carrying LR diplotypes presented a higher probability of 3-year TFS than those with HR diplotypes (68.5% vs 43.2%; p= 0.04). In regard to overall survival (OS), the estimated 5-year OS rates were 95.6% (95% CI 89-98) and 74.2% (95% CI 57-86) in the LR and HR group respectively (p= 0.005). Moreover, among the unmutated IGHV patients, those carrying LR diplotypes had a better 5-year OS compared to the patients with HR diplotypes (87.1% vs 71%; p= 0.02). Multivariate analysis demonstrated the IGHV mutation status (p= 0.005) and HLA-G diplotype-based risk groups (p= 0.01) to be independent factors predicting OS. Conclusions The results suggest the potential role of HLA-G and its polymorphisms in CLL. The inherited ability of the host to increase expression of the HLA-G antigen might contribute to the escape of CLL cells of the immuno-surveillance of the host and in turn to disease progression and the worse outcome for patients with CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2421 Background: Tumor necrosis factor (TNF)–α is an important pro-inflammatory cytokine involved in the modulation of lymphoma development and the balance between cell-mediated and humoral immunity. Deregulated concentrations of TNF–α have been detected in patients with lymphoma and were associated with an adverse prognosis. Evidence that the single-nucleotide polymorphism (SNP) in TNF promoter, TNF −308G〉A, could be the susceptibility locus for non-Hodgkin lymphoma (NHL) has been provided by case-control studies. Therefore we tested the hypothesis that TNF −308G〉A influences clinical course of B-cell chronic lymphocytic leukemia (CLL). Patients and Methods: We genotyped TNF −308G〉A (rs1800629) in 278 newly diagnosed patients with CLL and 192 ethnically-matched healthy individuals using the 7900 HT Real-Time (Applied Biosystems, USA). Some randomly selected DNA samples were analysed by direct sequencing using 3130xl Genetic Analyzer (Applied Biosystems, USA). Sequence data were based on the NCI SNP500 website. The IgVH mutation status in CLL patients was performed according to the protocol described by van Dongen et al. Serum samples from the 153 newly diagnosed CLL patients were collected at the time of diagnosis and tested by an enzyme-linked immunosorbent assay (ELISA) kit for human TNF (Quantikine, R&D Systems, USA). Results: The TNF −308G〉A allelic frequencies and distributions were consistent with Hardy-Weinberg equilibrium, and did not differ significantly between CLL patients and the control group. There were no significant differences between TNF allelic or genotype distributions and clinical characteristics of CLL patients at diagnosis, including age, clinical stage according to Rai classification, serum LDH and β2-microglobulin levels, surface CD38 expression, ZAP-70 expression, Döhner's cytogenetic groups, and IgVH mutation status. Neither of assessed TNF polymorphic variants was associated with response to first-line treatment, progression free survival (PFS) nor treatment free survival (TFS) that was measured from time point of diagnosis to first therapy. With a median follow-up of surviving patients of 52 months (range 1–209 months), the group of patients with TNF (−308A) allele (TNF−308AA or TNF−308AG genotypes) had significantly shorter overall survival (OS) compared to those carrying TNF (−308GG) genotype (p=0.01, log–rank test). To further characterize the prognostic impact of genetic variation in TNF on CLL patients survival, we divided the patients according to the IgVH mutation status into a IgVH unmutated (homology ≥98%) and a IgVH mutated (homology A polymorphism did not influence survival of patients with the IgVH mutated gene. To further investigate this difference, we analyzed the impact of TNF SNP on serum TNF-α levels in CLL patients at the time of diagnosis. We found that high TNF-α levels, greater than the median (16.84 pg/mL) value, correlated with stages III and IV according to Rai classification (p=0.001, chi2 test), elevated serum levels of LDH (p=0.01) or β2-microglobulin (p=0.001), CD38 expression ≥30% (p=0.001), ZAP-70 expression 〉20% (p=0.01) as well as with TNF (−308AA) genotypes (p=0.04). The patients with high TNF-α levels had significantly shorter TFS (p

Description: The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells. In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p

Description: Precursor B-cell lymphoblastic leukemia (B-ALL) in adults remains a challenging clinical problem due to higher relapse rate and worse prognosis than in children. Although most of adult patients respond to standard induction therapy and complete remissions (CR) are typically achieved in 90% of patients, the majority of them eventually relapse. Our previous studies indicate that the minimal residual disease (MRD) status after induction therapy is the most important risk factor of relapse in adult B-ALL patients [Br J Haematol 2008;142:227-37]. We hypothesized that the survival of B-ALL blasts after induction therapy is a result of intrinsic characteristics of the tumor cells that determine resistance to chemotherapeutics. Therefore, we sought to identify the molecular background of B-ALL cells resistance to daunorubicin. To identify potential mechanisms responsible for drug-resistant phenotype, we utilized gene expression data from previous studies that assessed transcriptional profiles of drug-sensitive and drug-resistant cells. Using gene set enrichment analysis (GSEA) of daunorubicin-sensitive versus -resistant phenotypes of B-ALL cells we identified differential expression HIF1α and MYC transcription factors target genes (p=0.002, FDR=0.144; p0.1%) MRD status after completion of the induction therapy. Among studied HIF1α and MYC targets, lactate dehydrogenase A (LDHA) expression was the best predictor differentiating MRD+ versus MRD- patients (p=0.0019, FDR=0.005). It was of particular interest, since tumor stem cells are typically characterized by MYC and HIF1α transcriptional signatures, which rewire cellular metabolism towards aerobic glycolysis. We next assessed the effect of LDHA inhibition with a small molecule inhibitor, GSK2837808A, on proliferation and clonogenicity of human B-ALL cell lines. GSK2837808A markedly reduced lactate production in B-ALL cell lines (RS4;11, SEM-K2 and NALM-6) and decreased proliferation and colony formation in semi-solid medium in a dose-dependent fashion. Taken together, we show that adult B-ALL patients with positive MRD status after induction therapy exhibit concordant upregulation of HIF1α and MYC signature genes. Expression of LDHA, a target gene regulated by both HIF1α and MYC transcription factors was significantly higher in MRD-positive patients. Finally, inhibition of LDHA markedly decreased proliferation and clonogenicity of B-ALL cell lines, indicating that LDHA might be a therapeutic target in B-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Chromosomal aberrations analyzed by cytogenetic and molecular methods are major prognostic factors determining treatment options in patients with acute leukemias. The aim of this study was to compare cytogenetic and molecular methods as diagnostic and prognostic tools in patients with acute leukemias. Sixty one previously untreated patients with acute leukemias (AML - 43, ALL - 18) were studied for the presence of chromosomal translocations and corresponding fusion genes [AML1-ETO t(8;21), CBFB-MYH11 inv(16), PML-RARA t(15;17), BCR-ABL p190, p210 t(9;22), MLL-AF4 t(4;11), TEL-AML1 t(12;21), E2A-PBX1 t(1;19), SIL-TAL1 del(1)] using both molecular (RT-PCR, nested PCR) and cytogenetic (GTG) methods. Molecular diagnostics was performed from RNA isolated from bone marrow samples according to BIOMED-1 protocol. Cytogenetic studies were carried out with classical GTG and FISH methods. G-banded mitoses of bone marrow specimen were analysed according to ISCN. Chromosomal aberrations were found in 32.8% patients using GTG method while the parallel molecular tests revealed related fusion genes in 50.8% patients. In 8.5% patients cytogenetic analysis was not performed because of lack of metaphases in cultured cells. All cytogenetic aberrations found in GTG were also confirmed in RT-PCR. Stratification into cytogenetic risk groups was performed after applying combined analysis of karyotyping and molecular tests. Low cytogenetic risk group consisted of 32.8% patients including 11.5% diagnosed with GTG and additionally 21.3% patients after applying molecular tests. The intermediate and high cytogenetic risk group consisted of 32.8% and 34.4% respectively using combined cytogenetic and molecular diagnostics. In low cytogenetic risk group, 85% of patients achieved complete remission (CR), early deaths were found in 15% and none of the patients presented primary chemotherapy resistance. In intermediate risk group CR were obtained in 80%, chemoresistance in 10% and early deaths were observed in 10% of patients. In high cytogenetic risk group, CR were achieved in only 23.8% and chemoresistance occured in 76.2% of the patients. In conclusion we suggest that molecular and cytogenetic tests are complementary methods and should be used in parallel in the initial diagnosis of patients with acute leukemias. This seems to be critical for obtaining the accurate diagnosis, cytogenetic risk assessment and choosing an optimal treatment options.

Description: Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells. We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform. We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors. Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells. PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway. Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy. Disclosures Warzocha: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.