Clonal Expansion Of Leukemic Cells Research Articles

Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL.Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure)Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively.Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data.To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens.To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response.Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes.Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. [Display omitted] DisclosuresLoughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

A Case of Acute Lymphocytic Leukaemia with t(3;13) and Central Nervous System Leukemia after Allogenic Cord Blood Transplantation

Background: Acute lymphoblastic leukemia (ALL) is a neoplastic cancer characterized by clonal expansion of leukemic cells in lymph organs and bone marrow. Lots of kinds of different chromosomal translocation can be found in those leukemic cells. However, the role of the abnormal chromosomals and genes in leukemogenesis is not yet fully understood. Identifying new chromosomal translocation can facilitate a better understanding of pathogenesis of this disease. Case presentation: We report a rare case of acute lymphocytic leukaemia with t(3;13)(q29,q21). The patient was diagnosed pre-B-ALL with no abnormal chromosomal or gene fusion and achieved complete remission (CR) after induction chemotherapy. Ten months later, she relapsed in the consolidation with cytogenetics test showing 46,XX,t(3;13)(q29,q21). Given no CR after 2 chemotherapy regimens, she received salvage cord blood transplantation. Regular intrathecal methotrexate was applied to prevent central nervous system leukemia. Good graft-versus-leukemia (GVL) effect was induced by daily injection of low dose of IL-2 two months post transplantation. Minimal residual disease (MRD) negativity was maintained until central nervous system leukemia was found 8 months after transplantation. A whole exome sequencing was performed. Nine driver mutation genes and seven tumor genes were found. Conclusions: We highly suspect that the relapse in central nervous system after transplantation is associated with the rare chromosomal translocation. Disclosures No relevant conflicts of interest to declare.

Multidisciplinary insight into clonal expansion of HTLV-1\u2013infected cells in adult T-cell leukemia via modeling by deterministic finite automata coupled with high-throughput sequencing

BackgroundClonal expansion of leukemic cells leads to onset of adult T-cell leukemia (ATL), an aggressive lymphoid malignancy with a very poor prognosis. Infection with human T-cell leukemia virus type-1 (HTLV-1) is the direct cause of ATL onset, and integration of HTLV-1 into the human genome is essential for clonal expansion of leukemic cells. Therefore, monitoring clonal expansion of HTLV-1–infected cells via isolation of integration sites assists in analyzing infected individuals from early infection to the final stage of ATL development. However, because of the complex nature of clonal expansion, the underlying mechanisms have yet to be clarified. Combining computational/mathematical modeling with experimental and clinical data of integration site–based clonality analysis derived from next generation sequencing technologies provides an appropriate strategy to achieve a better understanding of ATL development.MethodsAs a comprehensively interdisciplinary project, this study combined three main aspects: wet laboratory experiments, in silico analysis and empirical modeling.ResultsWe analyzed clinical samples from HTLV-1–infected individuals with a broad range of proviral loads using a high-throughput methodology that enables isolation of HTLV-1 integration sites and accurate measurement of the size of infected clones. We categorized clones into four size groups, “very small”, “small”, “big”, and “very big”, based on the patterns of clonal growth and observed clone sizes. We propose an empirical formal model based on deterministic finite state automata (DFA) analysis of real clinical samples to illustrate patterns of clonal expansion.ConclusionsThrough the developed model, we have translated biological data of clonal expansion into the formal language of mathematics and represented the observed clonality data with DFA. Our data suggest that combining experimental data (absolute size of clones) with DFA can describe the clonality status of patients. This kind of modeling provides a basic understanding as well as a unique perspective for clarifying the mechanisms of clonal expansion in ATL.

Cytokine-Mediated Inflammatory Pathways Promote Clonal Evolution and Disease Progression in Acute Myeloid Leukemia

Abstract Background: Inflammatory cytokines secreted in the bone marrow microenvironment play important roles in modulating cell survival, proliferation, differentiation, and immune responses in cancer. Perhaps not surprisingly, there is also an association between chronic inflammation and tumor progression. We recently used an ex vivo functional screen of 94 cytokines to show that the pro-inflammatory cytokines IL1α and IL1β promoted the expansion of AML progenitors in 70% (40/60) of primary samples. We therefore hypothesized that inflammatory cytokines are crucial to clonal expansion and disease progression in AML and that therapeutic targeting of these pathways may circumvent disease heterogeneity. Here we provide in vitro and in vivo evidence that IL1-mediated signaling elicits profound expansion of leukemia progenitors in AML patients harboring various genetic mutations and promotes in vivo clonal expansion and disease progression in a murine AML model. Further, these effects are reversed by targeting IL1 signaling. Methods: We validated the role of IL1 signaling using a shRNA approach and in murine competitive repopulation and bone marrow transplantation models. We evaluated the influence of these cytokines on inflammatory markers using immunoblotting, flow cytometry, and Luminex assays, and assessed strategies to target these pathways using small-molecule inhibitors. Results: IL1 stimulation promoted a 3- to 20-fold increase in growth, survival, and clonogenic potential of AML CD34+ cells, while paradoxically suppressing growth of healthy CD34+ cells. To identify the influence of IL1 on in vivo clonal expansion of healthy and leukemic progenitors simultaneously, we established an in vivo murine competitive repopulation study utilizing TET2-null mice. IL1 treatment promoted clonal expansion of TET2-null myeloid cells over wild-type cells during 6 weeks of IL1β treatment. Consistent with this, both flow cytometry analysis and blood differential counts showed an increased percentage of granulocytes and reduced percentage of lymphocytes in IL1-treated mice. In this model, TET2-null monocytes have greater expression of IL1 than wild-type cells, suggesting IL1 promotes clonal growth of TET2-mutated early leukemic progenitors. Similarly, IL1β and IL1 receptors (IL1R1 and IL1RAP) were overexpressed in IL1-sensitive AML bone marrow and peripheral blood samples compared to nonsensitive AML samples and normal samples. Intracellular FACS showed that the majority of IL1β was secreted by monocytes and to some extent by myeloid progenitors. Accordingly, IL1-sensitive AML samples exhibited trends towards monocytic and myelomonocytic clinical features. Reduced survival of AML cells after monocyte depletion was rescued by IL1 treatment, suggesting that IL1 mediates paracrine regulation of AML cell growth. Silencing of the IL1 receptor, IL1R1, reduced the clonogenic potential of AML primary samples and oncogene (AML1-ETO9a, NRASG12D, and MLL-ENL)-transduced mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer than did recipients of wild-type marrow. We also found that IL1β increased phosphorylation of p38MAPK and MK2, as well as secretion of multiple downstream inflammatory cytokines (IL6, IL8, MCP1, MIP1α, and MIP1β) from CD34+ progenitors, in IL1-sensitive AML samples compared to IL1-nonsensitive progenitors. Conversely, treating AML cells with p38MAPK inhibitors such as doramapimod andralimetinib reduced the growth of AML cells by decreasing p38MAPK and MK2 phosphorylation and reducing secretion of inflammatory cytokines from AML progenitors. Clinical and demographic analyses suggest that AML patients are dependent on IL1 signaling irrespective of mutation status and clinical features. Targeting this unifying mechanism of IL1-mediated clonal expansion may thus have application across heterogeneous AML subtypes. Conclusion: We demonstrate that IL1 promotes in vitro and in vivo clonal expansion of leukemic cells and promotes disease progression in AML. As IL1 signaling is active across heterogeneous disease subtypes, AML patients may therefore benefit from drugs targeting IL1/p38MAPK signaling because of their potential to inhibit AML while enhancing normal hematopoiesis, a significant clinical advantage over traditional chemotherapy. Disclosures Agarwal: CTI BioPharma Corp: Research Funding.

BCR-ABL truncation due to premature translation termination as a mechanism of resistance to kinase inhibitors

7028 Background: The major mechanism underlying imatinib resistance in patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with point mutations in the BCR-ABL tyrosine kinase. We describe three novel ABL premature termination mutations leading to BCR-ABL truncation in leukemia patients with multidrug (imatinib/nilotinib/dasatinib) resistance. Methods: Peripheral blood or bone marrow samples from drug-resistant CML patients were collected. Total nucleic acids were purified and subjected to two rounds of PCR analysis, with the first PCR designed to eliminate amplification of the wild-type, non-translocated ABL gene. Bi-directional sequencing was then performed. HL60 cells (a Ph-negative myeloid leukemia cell line) and peripheral blood of healthy subjects were used as negative controls; a human CML cell line (K562) was used as a positive control. Results: We identified an exon 7 deletion in three CML patients, a 4-nt insertion (908insCAGG) near the exon 5/6 junction in one CML case, and an exon 6 point mutation (997C>T) in one patient with acute lymphoblastic leukemia (ALL). These mutations all create premature stop codons and cause termination at residues 381, 315, and 333, respectively, leading to truncated proteins with only the first quarter of the kinase domain (P-loop) or lacking the C-terminus of ABL including the A-loop. Conclusions: These novel mutations, and the previously documented 35-nt insertion in exon 8, may constitute a new class of mutations that 1) cause truncation of the BCR-ABL kinase; (2) abolish the regulatory element in the ABL kinase domain and the downstream C-terminal region; and (3) confer significant drug resistance. [Table: see text]

A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloid leukemia–like syndrome in BXH-2 mice

BXH-2 mice develop a fatal myeloid leukemia by a two-step mutagenic process. First, a BXH-2–specific recessive mutation causes a myeloproliferative syndrome. Second, retroviral insertions alter oncogenes or tumor suppressors, resulting in clonal expansion of leukemic cells. We have identified a recessive locus on chromosome 8 (Myls) that is responsible for myeloproliferation in BXH-2. This Myls interval has been narrowed down to 2 Mb and found to contain several positional candidates, including the interferon consensus sequence–binding protein 1 gene (Icsbp, also known as interferon regulatory factor 8 [IRF8]). We show that BXH-2 mice carry a mutation (915 C to T) resulting in an arginine-to-cysteine substitution at position 294 within the predicted IRF association domain of the protein. Although expression of Icsbp1 mRNA transcripts is normal in BXH-2 splenocytes, these cells are unable to produce interleukin 12 and interferon-γ in response to activating stimuli, confirming that R294C behaves as a loss-of-function mutation. Myeloproliferation in BXH-2 mice is concomitant to increased susceptibility to Mycobacterium bovis (BCG) despite the presence of resistance alleles at the Nramp1 locus. These results suggest a two-step model for chronic myeloid leukemia in BXH-2, in which inactivation of Icsbp1 predisposes to myeloproliferation and immunodeficiency. This event is required for retroviral replication, and subsequent insertional mutagenesis that causes leukemia in BXH-2 mice.

Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants

Resistance is a major challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). We investigated the mechanisms of resistance in patients with progressive GISTs with primary KIT mutations and the efficacy of the kinase inhibitor PKC412 for the inhibition of imatinib-resistant mutants. We performed a cytogenetic analysis and screened for mutations of the KIT and PDGFRA kinase domains in 26 resistant GISTs. KIT autophosphorylation status was assessed by Western immunoblotting. Imatinib-resistant GIST cells and Ba/F3 cells expressing these mutant proteins were tested for sensitivity to imatinib and PKC412. Six distinct secondary mutations in KIT were detected in 12 progressive tumors, with V654A and T670I found to be recurrent. One progressive tumor showed acquired PDGFRA -D842V mutation. Amplification of KIT or KIT / PDGFRA was found in 2 patients. Eight of 10 progressive tumors available for analysis showed phosphorylated KIT. Two remaining progressive tumors lost KIT protein expression. GIST cells carrying KIT -del557-558/T670I or KIT -InsAY502-503/V654A mutations were resistant to imatinib, while PKC412 significantly inhibited autophosporylation of these mutants. Resistance to imatinib and sensitivity to PKC412 of KIT -T670I and PDGFRA -D842V mutants was confirmed using Ba/F3 cells. This study shows the high frequency of KIT/PDGFRA kinase domain mutations in patients with secondary resistance and defines genomic amplification of KIT / PDGFRA as an alternative cause of resistance to the drug. In a subset of patients, cancer cells lost their dependence on the targeted tyrosine kinase. Our findings show the sensitivity of the imatinib-resistant KIT -T670I and KIT -V654A and of PDGFRA -D842V mutants to PKC412.

Proliferation and apoptosis in acute and chronic leukemias and myelodysplastic syndrome

Clonal expansion of leukemic cells is thought to be due to proliferation in excess of apoptosis. To define and compare proliferation and apoptosis between various leukemias and myelodysplastic syndrome (MDS), we measured proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation as surrogate markers for proliferation and caspase 3 activity and annexin V surface binding as surrogate markers for activation of the apoptotic cascade in patients with MDS, chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). We found high proliferation in bone marrow cells from MDS and CMML as measured by PCNA and BrdU incorporation. The lowest level of proliferation was found in CLL. Apoptosis was also highest in MDS and CMML as measured by annexin V and caspase 3 activity. Unexpectedly, we found no significant difference in proliferation in bone marrow CD34+ cells from various leukemias or MDS. Apoptosis was significantly higher in bone marrow CD34+ cells from MDS and CML in chronic phase as compared to CD34+ cells from AML patients. Our results illustrate differences in proliferation and apoptosis between acute and chronic leukemias and MDS. These differences may have diagnostic and therapeutic implications.

Generation of a novel Fli-1 protein by gene targeting leads to a defect in thymus development and a delay in Friend virus-induced erythroleukemia.

The proto-oncogene Fli-1 is a member of the ets family of transcription factor genes. Its activation by either chromosomal translocation or proviral insertion leads to Ewing's sarcoma in humans or erythroleukemia in mice, respectively, Fli-1 is preferentially expressed in hematopoietic and endothelial cells. This expression pattern resembled that of c-ets-1, another ets gene closely related and physically linked to Fli-1. We also generated a germ line mutation in Fli-1 by homologous recombination in embryonic stem cells. Homozygous mutant mice exhibit thymic hypocellularity which is not related to a defect in a specific subpopulation of thymocytes or to increased apoptosis, suggesting that Fli-1 is an important regulator of a prethymic T-cell progenitor. This phenotype was corrected by crossing the Fli-1 deficient mice expressing Fli-1 cDNA. Homozygous mutant mice remained susceptible to erythroleukemia induction by Friend murine leukemia virus, although the latency period was significantly increased. Surprisingly, the mutant Fli-1 allele was still a target for Friend murine leukemia virus integration, and leukemic spleens with a rearranged Fli-1 gene expressed a truncated Fli-1 protein that appears to arise from an internal translation initiation site and alternative splicing around the neo cassette used in the gene targeting. The fortuitous discovery of the mutant Fli-1 protein, revealed only as the result of the clonal expansion of leukemic cells harboring a rearranged Fli-1 gene, suggests caution in the interpretation of gene-targeting experiments that result in either no or only a subtle phenotypic alteration.