Clones Of Leukemic Cells Research Articles

CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.

6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.

Simultaneous Occurrence Of Multiple Myeloma and Acute Myeloid Leukemia: A Dual Malignancy Of The Hematopoietic System

The occurrence of acute myeloid leukemia (AML) after the treatment for multiple myeloma (MM) is well described in several cases. The simultaneous presentation of MM and AML is very rare, with only 17 cases reported in articles found at PubMed.A 69 years old woman was sent at our institution for consultation due to pancitopenia at check-up. Past medical history was irrelevant. The CBC reported Hb 11.3 g/dL, Htc 33.8 %, ESR 32 mm/hr, Rouleaux formation +++, WBC 24.5 K/µL; differential showing nuetrophils 0.748 K/μL, lymphocytes 2.2 K/μL and myeloid blasts 20.1 K/µL, platelets 23.4 K/µL. The bone marrow aspiration was hypercellular with infiltration by myeloid blasts counting 95 %, and plasma cells with neoplastic morphology counting the remaining 5 %, no normal hematopoietic cells were observed. Hematological malignancy work up, also for MM, was performed, including flow cytometry (FC) and G band karyotype (GBK). FC revealed three populations: 1) representing 82.9 % that expressed CD11b +, CD13 ++, CD33 +, CD34 +, CD 38 +, HLA-DR + and MPO +; 2) representing 4.6 % and expressed CD38 +++, CD56 +, CD138 +++, kappa ++; 3) mixed population of normal B and T cells, representing 12.5 %. GBK showed 18 normal metaphases and 2 metaphases with t(11:14) (q13:q32) and del(13q). Stratification tests reported creatinin 2.3 mg/dL, , Ca 10.15 mg/dL, us-CRP 23 mg/dL, β-2-microglobulin 7.751 mg/L, albumin 2.35 g/dL, globulin 5.75 g/dL, serum protein electrophoresis with M-spike in the gamma region with 4.12 g/dL, positive Bence Jones protein, X-ray from the skull showed multiple lytic lesions. High risk MM and AML M4 were diagnosed. The patient did not accept any treatment and died few days later due a pulmonary hemorrhage.There is no pathophysiologic mechanism that explains the simultaneous occurrence. MM is a slowly disease, it may lead to chromosomal instability and immunosuppression with failure to eliminate clones of leukemic cells. No effective treatment is currently available, allogeneic stem cell transplantation is the best option. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Next-Generation Sequencing in Non-BCR-ABLMechanisms Resistance in Patients with Chronic Myeloid Leukemia: Results of a Pilot Study

Introduction.In recent times, more and more data are available on the mechanisms of development of primary and secondary resistance to therapy with tyrosine kinase inhibitors (TKIs) in patients with CML. The use of next-generation sequencing (NGS) allows detecting mutations in theBCR-ABLkinase domain (KD) in some patients with resistance to TKI therapy. However, the reasons for resistance in patients without mutations in theBCR-ABLKD are still unclear. Aim.To determineBCR-ABL-independent molecular genetic markers of resistance to TKI therapy in patients with CML using NGS. Materials and methods.We examined blood samples from 15 patients with resistant CP CML (8 men and 7 women) aged 32 to 59 years (Me = 44 years). Resistance to TKI therapy was determined according to the ELN 2013 criteria. In this group: 2 patients received therapy with 2 TKIs, 8 patients with 3 TKIs, 3 patients - 4 TKIs, and 2 patients received 5 TKIs (Me = 3 TKIs). Cytogenetic test revealed trisomy 8 in 3 patients. One patient was diagnosed with the T315I mutation at once. The patients underwent NGS examination of the myeloid panel, which included 55 genes, with an average reading depth of 1000x using a MiSeq device (Illumina). The 2% threshold of variant allele frequency (VAF) was used. The clinical significance of mutations was evaluated using the COSMIC, ClinVar and OMIM databases. Results.By NGS we identified genetic aberrations in all patients: an average of 5 mutations (from 1 to 10 in one patient). A total of 77 mutations with unclear clinical significance were identified: 54 of them were missense mutations, 22 were synonymous mutations, and 1 frameshift mutation. The most common abnormalities were found in theNF1(10),TET2(8),ATRX(7), andSTAG2(6) genes. In 2 patients, the simultaneous presence of mutations in theATRXandNF1genes was found, they had a primary-resistant course of the disease, MMR was not achieved even on the 3rd and 4th lines of targeted therapy. Three patients were found to have simultaneous mutations in theNF1andSTAG2genes, including two patients who also had a primary-resistant course of the disease, and one of them showed resistance to all 5 TKIs. In 2 patients with the simultaneous presence of mutations in theATRXandTET2genes, resistance to 2 TKIs was registered; CCyR without MMR was achieved only with ponatinib. In 3 patients with singleSTAG2mutations, MMR was achieved on the third line of TKI. One patient with 3 mutations inTET2also achieved MMR only on the third line TKI. Mutations inTET2are more common in Ph-negative MPNs. In our study, mutations were most often detected in theNF1,ATRXandSTAG2genes. Although mutations in theNF1andATRXgenes have been described previously, they were found in responding patients with CP CML (Kim et. al. Blood 2017). In our study, mutations in these genes were found in therapy-resistant patients with an unfavorable course of the disease. TheNF1gene encodes a GTPase activating protein (GAP), which is involved in inhibiting the RAS/MAPK signal transduction pathway and is a tumor suppressor. Mutations in this gene are often associated with the development of CMML and JMML, resistance to therapy and decreased overall survival. TheATRXgene encodes a protein involved in chromatin remodeling, regulation of transcription, replication, and maintenance of telomere structure. TheSTAG2gene encodes a protein that is part of the cohesin complex. Possibly, abnormalities in these genes may belong to a clone of leukemic stem cells (Vetrie et. al. Nature 2020), cause their resistance to TKI,BCR-ABL-independent proliferative potential and their genetic instability. In addition, some of these mutations, possibly, are preleukemic - they could precede the emergence of the Ph-clone in such patients by the mechanism of clonal hematopoiesis of indeterminate potential and may cause an unfavorable prognosis. This allowed us to suggest the prognostic and therapeutic value of mutations in theNF1,ATRXandSTAG2genes in CP CML patients with resistance to TKI therapy. Conclusion.The obtained results of a pilot study of NGS use for predictingBCR-ABL-independent resistance to TKI therapy in patients with CML can serve as a basis for further research aimed at developing prognostic models and choosing a treatment method for resistance to targeted therapy. Disclosures Voloshin: Novartis:Honoraria, Speakers Bureau.Fominykh:Novartis:Honoraria, Speakers Bureau;Pfizer:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau.Shuvaev:Novartis:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau;Pfizer:Honoraria, Speakers Bureau.Martynkevich:Pfizer:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau;Novartis:Honoraria, Speakers Bureau.

Mutations in PIGA cause a CD52-/GPI-anchor-deficient phenotype complicating alemtuzumab treatment in T-cell prolymphocytic leukemia.

Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing for PIGA mutational analyses. We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of the PIGA gene. One patient with relapsed T-PLL exhibited a single PIGA mutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three different PIGA mutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealed PIGA wild-type sequence reads in all 16 patients with samples available for testing. These data indicate that PIGA mutations were acquired during or after completion of alemtuzumab treatment.

Gene Editing of BTK in Acute Myeloid Leukaemia Using CRISPR-Cas9

Acute myeloid leukemia (AML) blood cancer is found primarily in adults and the elderly, being the third biggest blood cancer killer. In the last decades there has been some improved rates in younger patients, but little improvement in older patients. AML survival rates are the worst of all blood cancers, therefore finding new therapeutic targets to treat AML is crucial.Bruton's tyrosine kinase (BTK) is involved in the signalling of multiple receptors including growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen receptors and integrins. BTK in turn activates many of the major downstream signalling pathways that control cell migration, adhesion, survival and proliferation. BTK is best known for its role in B-cell receptor signalling, but BTK is also expressed and constitutively active in AML cell lines and AML patient blasts. Targeting BTK in B-cell malignancies with the BTK inhibitor ibrutinib has shown clinical effectiveness and tolerability in recent patient trials.Ibrutinib is orally active and covalently binds at the Cys-481 in the ATP-binding domain of BTK, to irreversibly block its enzymatic activity. A proportion of CLL and MCL patients treated with ibrutinib relapse during the therapy, with secondary resistance developing against ibrutinib. This resistance is mainly due to a BTK cysteine to serine mutation at position 481 (C481S) which disrupts ibrutinib's covalent binding to BTK.As little is known about BTK's role and therapeutic potential in AML, we aimed to study the effects and functional consequences of pharmacological inhibition of BTK using ibrutinib. We furthermore generated the BTK-C481S mutation in AML cell lines using the CRISPR-Cas9 gene-editing system. Shown here for the first time is a gene-edited mutation introduced into a human leukemia cell clone that has been purified to homogeneity. We explored the functional impact of BTK-C481S mutation on ibrutinib sensitivity, and mutation-induced adaptation of the phosphoproteome and kinome in AML cells.HEK-293T cells were used for validation purposes to find the most efficient knock-in strategy. We tested the most commonly used CRISPR endonuclease spCas9 as well as two other Cas9 orthologs called SaCas9 and AsCas12a. The designed spacer sequences within the sgRNA that target the gene of interest were delivered either via plasmid or in vitro transcripts. A repair template for homology directed repair (HDR) was designed containing the necessary base changes to change the amino acid at position 481 in BTK from cysteine to serine. The HDR template was delivered via transfection as single-stranded oligodeoxynucleotide (ssODN) or incorporated into plasmid. All strategies tested were successful but due to the poor transfectability of AML cell lines, we used lentiviral delivery to introduce BTK-C481S mutation into AML cell lines.The BTK-C481S mutation was introduced in THP-1 and OCI-AML3 cells. Mutant-positive cells were found via clonal selection and Sanger sequencing. Wild-type (WT) as well as mutant cells were treated with various ibrutinib concentrations ranging from 1 nM to 1 µM and analysed for BTK expression/phosphorylation via Western blot, with effects on cell proliferation, viability and cell cycle measured via flow cytometry. We also performed phosphoproteomics and kinome assays comparing untreated as well as treated (30 nM ibrutinib, 2h) THP-1 WT and THP-1 BTK-C481S cells.Western blot results showed that the effective concentration required for BTK inhibition is 30-fold higher in BTK-C481S mutant compared to WT cells (300 nM vs 10 nM). Wash-out experiments revealed that ibrutinib binding can be reversed in BTK-C481S mutant cells but not WT cells. Little effect of ibrutinib on proliferation and viability were seen comparing BTK-C481S mutant and WT AML cells.These signalling adaptations in BTK-C481S cells are consistent with changes observed in ibrutinib-resistant leukemia patient samples. DisclosuresSlupsky:Verastem: Research Funding.

Use of MRD for Selection of Post Remission Therapy in Adult All Management

After frontline induction therapy, the complete remission (CR) rate is achieved in around 90% of newly diagnosed adults with ALL, irrespectively of the regimen administered (BFM-derived or HyperCVAD-based). However, relapse remains the main cause of treatment failure in adults with ALL, and half of the adults who achieve CR will relapse. Thus, CR is not a good surrogate marker for long-term outcomes in ALL. Minimal residual disease (MRD) refers to the ability to detect and quantify small residual clones of leukemic cells not detected by morphology at any time during or after the treatment of ALL.1 MRD has abrogated most of the other conventional prognostic features in ALL, and is being increasingly used to guide subsequent therapy after induction and consolidation, especially to decide whether to perform allogeneic hematopoietic stem cell transplantation (alloHSCT) or to better design rational post-remission therapies using novel agents or procedures.2

Epigenetic dysregulation of the erythropoietic transcription factor KLF1 and the β-like globin locus in juvenile myelomonocytic leukemia

ABSTRACTIncreased levels of fetal hemoglobin (HbF) are a hallmark of more than half of the children diagnosed with juvenile myelomonocytic leukemia (JMML). Elevated HbF levels in JMML are associated with DNA hypermethylation of distinct gene promoter regions in leukemic cells. Since the regulation of globin gene transcription is known to be under epigenetic control, we set out to study the relation of DNA methylation patterns at β-/γ-globin promoters, mRNA and protein expression of globins, and epigenetic modifications of genes encoding the globin-regulatory transcription factors BCL11A and KLF1 in nucleated erythropoietic precursor cells of patients with JMML. We describe several altered epigenetic components resulting in disordered globin synthesis in JMML. We identify a cis-regulatory upstream KLF1 enhancer sequence as highly sensitive to DNA methylation and frequently hypermethylated in JMML. The data indicate that the dysregulation of β-like globin genes is a genuine attribute of the leukemic cell clone in JMML and involves mechanisms not taking part in the normal fetal-to-adult hemoglobin switch.

A novel AHI-1\u2013BCR-ABL\u2013DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

Tyrosine kinase inhibitor (TKI) therapies induce clinical remission with remarkable effects on chronic myeloid leukemia (CML). However, very few TKIs completely eradicate the leukemic clone and persistence of leukemic stem cells (LSCs) remains challenging, warranting new, distinct targets for improved treatments. We demonstrated that the scaffold protein AHI-1 is highly deregulated in LSCs and interacts with multiple proteins, including Dynamin-2 (DNM2), to mediate TKI-resistance of LSCs. We have now demonstrated that the SH3 domain of AHI-1 and the proline rich domain of DNM2 are mainly responsible for this interaction. DNM2 expression was significantly increased in CML stem/progenitor cells; knockdown of DNM2 greatly impaired their survival and sensitized them to TKI treatments. Importantly, a new AHI-1–BCR-ABL–DNM2 protein complex was uncovered, which regulates leukemic properties of these cells through a unique mechanism of cellular endocytosis and ROS-mediated autophagy. Thus, targeting this complex may facilitate eradication of LSCs for curative therapies.

Therapy - Related Myeloid Neoplasm in a Patient with Prior Treatment for Chronic Lymphocytic Leukemia: A Case Report

Background: Acute myeloid leukemia or myelodysplastic syndrome, during the course of chronic lymphocytic leukemia, is a rare entity. In such a situation, most previous cases were therapy-related after treatment. However, in very rare cases, acute myeloid leukemia has been diagnosed in untreated patients with chronic lymphocytic leukemia. Materials and Methods: In this paper, we present the case-study of a 60-year old woman who was diagnosed six years previously as being afflicted with chronic lymphocytic leukemia with no chromosomal changes. She was successfully treated with fludarabine in combination with cyclophosphamide and rituximab, and she obtained complete remission, yet with incomplete recovery. Subsequently, three years later, she was re-admitted to hospital with deep pancytopenia. Her bone marrow biopsy then revealed that 52% of her blasts cells demonstrated a myelomonocytic immunophenotype, albeit with no chronic lymphocytic leukemia cell clone, showing the following complex chromosomal abnormalities: 45~47, XX, del(5)(q13q33), trisomy 8, trisomy 20, monosomy 21, +mar[cp12]/46, XX. Thus, a diagnosis was made of acute myelomonocytic leukemia preceded by a transforming myelodysplastic syndrome. The patient received induction chemotherapy and obtained complete remission. Unfortunately, one month after completion of her consolidation regimen, a relapse occurred. She died 8 months post-diagnosis of AML. Conclusion: In this case study, we diagnosed therapy-related acute myeloid leukemia. Of note, the theory of a common stem cell malignancy and the coexistence of two malignant clones seem not to be applied in the presented case. This is because an acute myeloid leukemia clone was not seen in her bone marrow, nor was peripheral blood evidenced at the time of her diagnosis of chronic lymphocytic leukemia.

Telomere Length Shortening in the CML Leukemic Stem Cell Compartment Correlates with the Respective Clone Size: Evidence for Early Vs. Late Chronic Phase

Introduction: Chronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the bcr-abl translocation. Recent data provides evidence that CML chronic phase (CP) can be classified into early and late CP depending on the degree of expansion of the leukemic stem cell (LSC) clone. Patients in late CP have a higher LSC burden going along with an inferior response to TKI therapy. Telomeres shorten with each cell division and telomere length (TL) reflects the replicative history of a cell. We postulate that the LSC burden correlates with accelerated telomere shortening due to clonal replicative expansion of bcr-abl positive cells. Previous studies evaluating TL in peripheral blood cells of CML patients already revealed a correlation of age-adapted TL with disease stage, response to treatment and duration of CP. However, the high intra-individual, mostly genetic inter-individual variability in TL limits the predictive value of TL measurements when no patient specific bcr-abl negative cells were available for comparison. The aim of our study was to analyze TL in the LSC and non-clonal HSC compartment of patients with CP using a modified Q-FISH technique allowing the TL analysis in bcr-abl positive and negative cells.Methods and Patients: 11 patients diagnosed with CML in CP of the NCT00852566 study (Nordic CML Group) were included into our retrospective analysis. Mean age of the patients was 58.9 years. Bone marrow samples from initial diagnosis were sorted for CD34+/CD38- cells reflecting the leukemic stem cell compartment. Samples were analyzed with the FISH method using dual fusion dual color BCR-ABL1 probe following standard procedures. After capturing the bcr-abl staining using confocal microscopy, samples were re-processed for TL analysis by Q-FISH using established protocols. Optimized microscopy techniques allowed for TL to be assessed in all previously captured cells allowing the identification of bcr-abl positive and negative cells within the same sample. Analysis and quantification of bcr-abl FISH staining and TL measurement by Q-FISH were performed in single-blinded fashion.Results: Bcr-abl negative cells represent the non-clonal hematopoiesis and were used to correct TL. We observed significant shortened TL in the bcr-abl positive cells compared to bcr-abl negative cells (-2.18 ± 2.08 kilobases (kb), p=0.01) in line with previously published results. Next, we correlated the clone size (i.e. the proportion of bcr-abl positive cells) with the degree of telomere shortening in the leukemic stem cell compartment. Mean clone size of the patients was 67.4 ± 21.7 % S.D. Despite of the relatively small sample size studied, we found a significant negative correlation (R²=0.45, p=0.04) between TL and clone size strongly supporting the notion that increased expansion of the bcr-abl positive LSC pool leads to accelerated telomere shortening.Conclusions: In this study, we provide evidence for accelerated telomere shortening in bcr-abl positive LSC as compared to their normal CD34+/CD38- counterpart in CP CML samples at diagnosis. Furthermore, the degree of TL shortening correlates with the clone size in the HSC compartment, i.e. a parameter that reflects duration of CP. Thus, this allows the discrimination of early vs. late CP and might as such be used as a prognostic (and potentially predictive) biomarker in CML. DisclosuresRichter:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Fioretos:Cantargia: Equity Ownership. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Brümmendorf:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties.

A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells.

The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors.

TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality.

Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.

Bifurcation patterns in generalized models for the dynamics of normal and leukemic stem cells with signaling

The dynamics of normal and leukemic stem cell clones competing for resources in the same bone marrow niche can be modeled in a systems biology approach. We derive three related models describing the time evolution of normal and chronic myelogenous leukemia (CML) stem cells. The approach is based on the idea that stem cell proliferation is regulated by feedback mechanisms. We assume here that the growth of stem cell clones can be approximated by Tsallis functions, which depend on population numbers to ensure the communication between different stem cell strains. Of particular interest is the influence of medication, for example, by the CML drug imatinib, on the stem cell dynamics, which can be simulated by a variation of the parameterization of the differential equation systems. The basic 2D model represents the contest between cycling normal and wild‐type CML stem cells. In addition, extension of the basic model (i) by coupled reservoirs of quiescent stem cells and (ii) by a third stem cell species, which could be interpreted as an imatinib‐resistant mutant, is investigated. We analyze the global dynamics of the corresponding 2D, 3D, and 4D equation systems by analytic means and provide a complete map of the bifurcation landscape that occurs for changing values of the respective signaling strengths and growth or death parameters. This includes a complete classification of equilibria and their linear and non‐linear Lyapunov stability. Copyright © 2014 John Wiley & Sons, Ltd.

Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

Genomic Characterization Of Histiocytic Lesions Following Pediatric T-Cell Acute Lymphoblastic Leukemia

IntroductionThe development of histiocytic lesions after acute lymphoblastic leukemia (ALL) is rare, occurring in 6 of 971 patients enrolled on BFM treatment regimens for T-ALL (Trebo 2005). Few cases of histiocytosis arising after a history of T-ALL have been characterized at the molecular level for genomic alterations. One case of fatal Langerhans cell histiocytosis (LCH) following treatment of T-ALL revealed activating mutations in NOTCH1; in contrast, no NOTCH1 mutations were identified in 24 other cases of LCH or Rosai-Dorfman disease without a previous history of T-ALL (Rodig 2008). In patients without prior leukemia, BRAF V600 mutations have been identified in a significant proportion of patients with LCH or Erdheim-Chester Disease but not in other histiocytoses (Haroche 2012). Methodology and Principal FindingsNext generation focused exomic sequencing of 236 genes and 47 Introns was conducted on samples of histiocytic lesions from two patients with a previous history of T-ALL. Case 1A 2 year-old male presented with marked adenopathy, mediastinal mass and white blood cell count 67,000 cells/uL with 30% blasts. The blasts expressed CD45, CD2, CD3 (surface/cyto), CD5, CD7, CD38, CD45 (bright), TCR gamma-delta, but were negative for CD4, CD8, and TdT. Karyotype was 46,XY,t(8;14)(q24;q11.2),der(12)t(12;20)(q11;q13.3),der(20)t(12;20)(q21;q13.3) with rearrangement of MYC (8q24) confirmed by FISH. He was treated for T-ALL per Children's Oncology Group (COG) protocol AALL0434 and had 5% residual bone marrow blasts at day 29 of induction. MRD-negative remission was ultimately achieved with high-dose cytarabine and methotrexate followed by consolidation with nelarabine. He underwent matched unrelated cord blood transplant following conditioning with cyclophosphamide and total body irradiation with cranial boost, and engrafted at day +14. Surveillance bone marrow at day +110 revealed systemic juvenile xanthogranuloma (JXG) without T-ALL. PET/CT revealed FDG-uptake in the diffusely enlarged spleen and throughout the skeleton. Due to progressive cytopenias, therapy was initiated with vinblastine and prednisone as per LCHIII. However, refractory cytopenias exacerbated by splenic sequestration developed following induction, and he was then treated with thalidomide and splenectomy. The spleen weighed 404 grams (expected 40 grams) and was diffusely infiltrated with JXG. The cytopenias dramatically improved and he continued thalidomide for 2 months until PET scan demonstrated progression. Genomic analysis of the JXG lesion revealed NRAS G13D mutation, and FISH demonstrated MYC rearrangement identical to the initial T-ALL sample. Case 2A 12-year-old male presented with WBC 142,700 cells/uL and CNS leukemia. Flow cytometry showed T-ALL with CD2, surface CD3, CD4, CD5, CD7, CD8, CD24 (subset), CD71, HLA-DR (subset) and TdT (partial). There was a clonal TCR gamma gene rearrangement and a biallelic CDKN2A (p16) deletion by FISH. He was enrolled on COG AALL0434 and had a rapid response with remission in both CNS and marrow at induction day 29. Following completion of high-dose methotrexate interim maintenance he developed hepatosplenomegaly, pancytopenia and elevated serum bilirubin, ferritin, and triglycerides. Bone marrow aspirate showed rare hemophagocytosis but no evidence of T-ALL. He was treated with dexamethasone and etoposide with no response. Follow-up bone marrow revealed brisk hemophagocytosis and a diffuse histiocytic neoplasm. Karyotype was 48,XY,+7,+11[2] /49,idem,+18[3] /46,XY[14]. PET/CT showed hepatosplenomegaly with FDG uptake in anterior mediastinum, hepatic nodules, spleen, and bone marrow. He was treated with Campath and then with intensive chemotherapy with fludarabine, cytarabine, and liposomal daunorubicin with no response and ultimately succumbed to disease. Genomic analysis of the clonal histiocytic infiltrate revealed KRAS G12C, BRAF G469V, NOTCH1 Q2440, and CCND2 G268R mutations, and FISH positive for biallelic CDKN2A (p16) deletion similar to original T-ALL. ConclusionsOur extensive genomic characterization suggests a unique molecular pathogenesis for histiocytic disorders arising after T-cell ALL and identified RAS signaling pathway and NOTCH1 mutations. Furthermore, these findings strongly indicate a potential derivation or trans differentiation from the malignant leukemic stem cell clone. Disclosures:No relevant conflicts of interest to declare.

Non-Linear Clonal Evolution Of Leukemia Driven By Selective Immune Pressure and Revealed By HLA Typing and High-Depth Exome Sequencing

IntroductionAllogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the best curative option for many patients with Acute Myeloid Leukemia and related malignancies, mostly due to the antileukemic activity of immune cells contained in the graft. Although in most of the cases HSCT can accomplish apparent disease eradication, frequently residual leukemic cells are able to evade elimination, eventually outgrowing and resulting in clinical relapse. Apparently indistinguishable from disease at diagnosis, relapses commonly display a more aggressive behavior, and most of the available therapeutic options are ineffective. Next-generation sequencing provides the opportunity to track specific alterations and disease subclones during the clinical history of patients, and from this information to identify mechanisms by which leukemic cells evade elimination. MethodsSerial disease samples collected longitudinally during the complex clinical history of a patient with high-risk myelodysplastic syndrome were analyzed by genomic HLA typing and by high-depth exome sequencing (minimum depth of coverage 70x). Patient fibroblasts and mononucleated peripheral blood cells harvested at remission served as reference for germline HLA typing and exome sequence. Whenever possible, leukemic blast were FACS-purified. Sequencing data were aligned to the reference genome using the BWA aligner and analyzed using the tools developed from the Cancer Genome Analysis group at the Broad institute to identify newly acquired mutations, clonal segregation of novel and pre-existing mutations and quantitative clonal dynamics. ResultsA 54 year old patient with high-risk refractory anemia with excess blasts (pancytopenia, blasts 19%, adverse cytogenetics) underwent unmanipulated T cell-repleted HLA-haploidentical HSCT in the presence of active disease. After one year of remission a first relapse occurred. HLA typing of the leukemic blasts harvested at relapse demonstrated the selective genomic loss of the mismatched HLA haplotype targeted by donor T cells, a frequent mechanism of leukemia immune evasion after haploidentical HSCT, first described by our group (Vago et al, N Engl J Med, 2009). The patient was re-transplanted from a different haploidentical donor mismatched for the HLA alleles retained by the mutated variants; accordingly, T cells from this donor were expectedly alloreactive against the relapsed leukemia. The patient re-obtained complete remission, which was maintained for five years, before the occurrence of a second relapse. Unexpectedly, at this relapse leukemic cells had lost the HLA molecules mismatched with the second donor, and expressed those that had gone amiss at first relapse. Since at both relapses HLA haplotype loss was due to a stable genomic alteration, chromosome 6p acquired uniparental disomy (aUPD), any linear relation between the first and second relapse could be ruled out (Figure 1). Leukemic samples at diagnosis, first relapse and second relapse were further characterized by high-depth exome sequencing, revealing that most of the mutations present in each of the three samples were not found in the other two: only a minute fraction of the mutations could be identified in all three disease presentations, possibly comprising the “driver mutations” harbored by the original leukemic stem cell clone. Amongst these shared mutations we are currently validating the functional and epidemiological relevance of missense alterations in the TP53 tumor suppressor gene, in the NHEJ1 gene (related to chromosomal instability), and in the BTNL8 gene, encoding the costimulatory receptor B7-H5, which may putatively play a role in leukemia recognition by T cells. ConclusionsOur results demonstrate that antileukemic immunity has a profound impact on leukemia clonal evolution, driving the selection of immuno-privileged subclones. Importantly, the genetic alterations shared between the leukemic variants that emerged at years of distance during the clinical history of our patient suggest the long-term persistence of a reservoir of leukemic stem cells which are resistant to, or in stable equilibrium with, the immune pressure that sculpted their progeny. Finally, the longitudinal approach adopted in this study holds promise for the identification of leukemia “driver” mutations, to provide new insights into leukemogenesis and new rationales for targeted eradicating therapies.▪ Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.

Alterations Of The Chemokine Microenvironment In Chronic Lymphocytic Leukemia

BackgroundIn chronic lymphocytic leukemia (CLL), proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T-cells and CD68+ nurselike cells (NLCs). In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T-cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined. MethodsIn order to address this issue, we measured serum levels of 20 chemokines and cytokines in a prospective cohort of 159 previously untreated Binet stage A patients (pts) (GCLLSG CLL1 trial), 50 pts with advanced CLL in need of first line treatment (GCLLSG CLL8 trial) and 27 healthy individuals. For the study cohorts, serum samples had been centrally collected at study entry and stored at -80°C. Sera were analyzed on a Luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters. ResultsSerum levels of 13 chemokines (EGF, MCP-1, MIP-1alpha, MIP-1beta, sIl2Ralpha, VEGF, MCP-2, MCP-4, SDF-1, 6CKine, CTACK, TRAIL, SCF) differed significantly between healthy controls and CLL patients (p<0.05), indicating that the expression of these cytokines may potentially impact pathways common to CLL and the disease specific microenvironment. In addition, 7 chemokine serum levels were significantly higher in advanced stage CLL compared to patients at primary diagnosis, indicating a pronounced shift of the chemokine homeostasis upon disease progression. (Table)TableMedian chemokine serum levels in healthy individuals and patients with early (CLL1 cohort) and advanced (CLL8 cohort) CLLMedian serum level (pg/ml)Healthy controlCLL1CLL8MIP-1beta116266196p<0.001sIl2Ralpha215042255p<0.001TNFalpha124080p=0.008Il-10319p=0.001IL-1655203425p=0.0136CKine319307764p<0.001SCF107955p=0.001We identified a strong correlation of inflammatory cytokines (TRAIL, TNFalpha; p=0.039), growth factors (EGF, VEGF, TPO, SCF; p<0.001) and chemokines involved in B- and T-cell migration and homing (MIP-1alpha, MIP-1beta, 6CKine, Il-16; p<0.05). These coordinately regulated cytokines may reflect pathways important to the pathobiology of CLL. Hierarchical cluster analyses revealed distinct chemokine expression patterns in healthy individuals, early CLL and advanced stage disease. Finally, sIl2Ralpha has been previously confirmed as an independent prognostic factor in early stage CLL ConclusionDue to the comparison of 27 healthy individuals and 209 CLL pts we were able to demonstrate that the chemokine homeostasis is significantly altered in CLL compared to healthy individuals. Chemokines involved in B- and T-cell migration and homing and recruitment, inflammation and growth factors are coordinately overexpressed, pointing towards pathways crucial to the formation of a disease specific microenvironment. These pathways might serve as potential targets for future therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

FANCA and FANCC modulate TLR and p38 MAPK–dependent expression of IL-1β in macrophages

AbstractHematopoietic stem and progenitor cells with inactivated Fanconi anemia (FA) genes, FANCA and FANCC, are hypersensitive to inflammatory cytokines. One of these, tumor necrosis factor α (TNF-α), is also overproduced by FA mononuclear phagocytes in response to certain Toll-like receptor (TLR) agonists, creating an autoinhibitory loop that may contribute to the pathogenesis of progressive bone marrow (BM) failure and selection of TNF-α–resistant leukemic stem cell clones. In macrophages, the TNF-α overproduction phenotype depends on p38 mitogen-activated protein kinase (MAPK), an enzyme also known to induce expression of other inflammatory cytokines, including interleukin 1β (IL-1β). Reasoning that IL-1β might be involved in a like autoinhibitory loop, we determined that (1) TLR activation of FANCA- and FANCC-deficient macrophages induced overproduction of both TNF-α and IL-1β in a p38-dependent manner; (2) exposure of Fancc-deficient BM progenitors to IL-1β potently suppressed the expansion of multipotent progenitor cells in vitro; and (3) although TNF-α overexpression in FA cells is controlled posttranscriptionally by the p38 substrate MAPKAPK-2, p38-dependent overproduction of IL-1β is controlled transcriptionally. We suggest that multiple inflammatory cytokines overproduced by FANCA- and FANCC-deficient mononuclear phagocytes may contribute to the progressive BM failure that characterizes FA, and that to achieve suppression of this proinflammatory state, p38 is a more promising molecular therapeutic target than either IL-1β or TNF-α alone.

Combined immunophenotyping and fluorescence in situ hybridization with chromosome-specific DNA probes allows quantification and differentiation of ex vivo generated dendritic cells, leukemia-derived dendritic cells and clonal leukemic cells in patients with acute myeloid leukemia

Antileukemic T-cell responses induced by leukemia-derived dendritic cells (DCleu) are variable, due to varying DC/DCleu composition/quality. We studied DC/DCleu composition/quality after blast culture in four DC media by flow cytometry (FC) and combined fluorescence in situ hybridization/immunophenotyping analysis (FISH-IPA). Both methods showed that DC methods produce variable proportions of DC subtypes. FISH-IPA is an elaborate method to study clonal aberrations in blast/DC cells on slides, however without preselection of distinct cell populations for FISH analysis. FISH-IPA data proved previous FC data: not every clonal/blast cell is converted to DCleu (resulting in various proportions of DCleu) and not every detectable DC is of clonal/leukemic origin. Preselection of the best of four DC methods for “best” DC/DCleu generation is necessary. DCleu proportions correlate with the antileukemic functionality of DC/DCleu-stimulated T-cells, thereby proving the necessity of studying the quality of DC/DCleu after culture. FC is the superior method to quantify DC/DCleu, since a blast phenotype is available in every given patient, even with low/no proportions of clonal aberrations, and can easily be used to study cellular compositions after DC culture.

Tracking molecular relapse of chronic myeloid leukemia by measuring Hedgehog signaling status

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a leukemic stem cell (LSC) clone, carring a Philadelphia translocation, able to overcome the non-malignant hematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are gold-standard for CML treatment. Each shows an impressive rate of complete cytogenetic response in chronic phase (CP)-CML. However, relapse and treatment failure are major problems with long-term use of TKIs. A polymerase chain reaction (PCR) assay to detect the mRNA expression of BCR–ABL1 represents the main molecular approach to monitoring response to treatment. However, using this analysis it is currently not possible to prospectively identify patients whose disease will relapse due to LSC reappearance. The aim of our study was to investigate whether the mRNA expression analysis of two Hedgehog (Hh) stemness signaling molecules, Smoothened (SMO) and Patched-1 (PTCH1), could predict upcoming molecular relapse. At the time of diagnosis, patients with high Sokal risk (n = 12) showed higher and lower levels of SMO and PTCH1, respectively (p = 0.0132), compared with patients with different Sokal scores (p = 0.0316 for intermediate risk and p = 0.0340 for low risk). These data suggest that Hh signaling was functionally more active in this risk group at the time of diagnosis. Furthermore, the kinetics of Hh signaling activity during the individual medical history correlated with BCR–ABL1 mRNA level and with upcoming molecular relapse. Also, mutation analysis of BCR–ABL1 revealed that activation of Hh signaling precedes molecular relapse by several months, mostly in patients carrying the gatekeeper mutation T315I. Importantly, in vitro data showed a synergistic effect of chemical inhibitors of Hh signaling and TKIs in both wild-type and resistant (T315I) CML cell lines. Collectively our data show that monitoring Hh pathway activity contemporaneously with BCR–ABL1 mRNA level may improve the chance of early detection of patients who will experience a relapse (mainly in the high Sokal risk group), paving the way for an innovative management of this hematologic malignancy.