Diverse and reprogrammable mechanisms of malignant cell transformation in lymphocytes: pathogenetic insights and translational implications.

While normal B- and T-lymphocytes require antigenic ligands to become activated via their B- and T-cell receptors (BCR and TCR, respectively), B- and T-cell lymphomas show the broad spectrum of cell activation mechanisms regarding their dependence on BCR or TCR signaling, including loss of such dependence. These mechanisms are generally better understood and characterized for B-cell than for T-cell lymphomas. While some lymphomas, particularly the indolent, low-grade ones remain antigen-driven, other retain dependence on activation of their antigen receptors seemingly in an antigen-independent manner with activating mutations of the receptors playing a role. A large group of lymphomas, however, displays complete antigen receptor independence, which can develop gradually, in a stepwise manner or abruptly, through involvement of powerful oncogenes. Whereas some of the lymphomas undergo activating mutations of genes encoding proteins involved in signaling cascades downstream of the antigen-receptors, others employ activation mechanisms capable of substituting for these BCR- or TCR-dependent signaling pathways, including reliance on signaling pathways physiologically activated by cytokines. Finally, lymphomas can develop cell-lineage infidelity and in the extreme cases drastically rewire their cell activation mechanisms and engage receptors and signaling pathways physiologically active in hematopoietic stem cells or non-lymphoid cells. Such profound reprograming may involve partial cell dedifferentiation or transdifferentiation towards histocytes, dendritic, or mesodermal cells with various degree of cell maturation along these lineages. In this review, we elaborate on these diverse pathogenic mechanisms underlying cell plasticity and signaling reprogramming as well as discuss the related diagnostic and therapeutic implications and challenges.

Genomic Evolution of Oligometastatic Clear Cell Renal Cell Carcinoma Presenting Two Decades Following Radical Nephrectomy.

Metachronous oligometastatic clear cell renal cell carcinoma may take many years before becoming clinically apparent. Herein we report regional lymph node recurrence of clear cell renal cell carcinoma more than two decades following radical nephrectomy. Chromosomal microarray analysis demonstrated multiple chromosomal alterations, including 3pq deletion shared by the original and recurrent tumors, and 17p deletion containing the TP53 gene present only in the latter. Sequencing of 1550 genes revealed mutations of VHL in both the primary and metastasis and BAP1 only in the metastatic lesion. These findings genetically link the original and recurrent tumors and suggest that VHL, TP53, and BAP1 alterations played an evolutionary role in recurrence decades after initial resection.

Validation of a Molecular Diagnostic Test for Circulating Tumor DNA by Next-Gen Sequencing.

A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

Chimeric kinase ALK induces expression of NAMPT and selectively depends on this metabolic enzyme to sustain its own oncogenic function.

As we show in this study, NAMPT, the key rate-limiting enzyme in the salvage pathway, one of the three known pathways involved in NAD synthesis, is selectively over-expressed in anaplastic T-cell lymphoma carrying oncogenic kinase NPM1::ALK (ALK + ALCL). NPM1::ALK induces expression of the NAMPT-encoding gene with STAT3 acting as transcriptional activator of the gene. Inhibition of NAMPT affects ALK + ALCL cells expression of numerous genes, many from the cell-signaling, metabolic, and apoptotic pathways. NAMPT inhibition also functionally impairs the key metabolic and signaling pathways, strikingly including enzymatic activity and, hence, oncogenic function of NPM1::ALK itself. Consequently, NAMPT inhibition induces cell death in vitro and suppresses ALK + ALCL tumor growth in vivo. These results indicate that NAMPT is a novel therapeutic target in ALK + ALCL and, possibly, other similar malignancies. Targeting metabolic pathways selectively activated by oncogenic kinases to which malignant cells become "addicted" may become a novel therapeutic approach to cancer, alternative or, more likely, complementary to direct inhibition of the kinase enzymatic domain. This potential therapy to simultaneously inhibit and metabolically "starve" oncogenic kinases may not only lead to higher response rates but also delay, or even prevent, development of drug resistance, frequently seen when kinase inhibitors are used as single agents.

Simultaneous Targeting of DNA Polymerase Theta and PARP1 or RAD52 Triggers Dual Synthetic Lethality in Homologous Recombination-Deficient Leukemia Cells.

DNA polymerase theta (Polθ, encoded by POLQ gene) plays an essential role in Polθ-mediated end-joining (TMEJ) of DNA double-strand breaks (DSB). Inhibition of Polθ is synthetic lethal in homologous recombination (HR)-deficient tumor cells. However, DSBs can be also repaired by PARP1 and RAD52-mediated mechanisms. Because leukemia cells accumulate spontaneous DSBs, we tested if simultaneous targeting of Polθ and PARP1 or RAD52 enhance the synthetic lethal effect in HR-deficient leukemia cells. Transformation potential of the oncogenes inducing BRCA1/2-deficiency (BCR-ABL1 and AML1-ETO) was severely limited in Polq-/-;Parp1-/- and Polq-/-;Rad52-/- cells when compared with single knockouts, which was associated with accumulation of DSBs. Small-molecule inhibitor of Polθ (Polθi) when combined with PARP or RAD52 inhibitors (PARPi, RAD52i) caused accumulation of DSBs and exerted increased effect against HR-deficient leukemia and myeloproliferative neoplasm cells. IMPLICATIONS: In conclusion, we show that PARPi or RAD52i might improve therapeutic effect of Polθi against HR-deficient leukemias.

Transdifferentiation, phenotypic infidelity, progression, and transformation in T/NK-cell neoplasms: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: Sessions 8 and 9 of the 2021 Society for Hematopathology and the European Association for Haematopathology Workshop aimed to collect examples of transdifferentiation, lineage infidelity, progression, and transformation in precursor and mature T/natural killer (NK)-cell neoplasms. METHODS: Twenty-eight cases were submitted and analyzed, with whole-exome sequencing and genome-wide RNA expression analysis performed in a subset of the cases. RESULTS: In session 8, 7 T-lymphoblastic lymphoma/leukemia cases were received that showed transdifferentiation to clonally related mature myeloid hematopoietic neoplasms, including 6 histiocytic/dendritic cell lineage neoplasms and a mast cell sarcoma. Session 9 included 21 mature T-cell neoplasms that were grouped into 3 themes. The first one addressed phenotypic infidelity in mature T-cell lymphomas (TCLs) and included 8 TCLs expressing aberrant antigens, mimicking classic Hodgkin and non-Hodgkin B-cell lymphomas. The second theme addressed disease progression in TCL and included 5 cutaneous T-cell lymphoproliferative disorders and 2 T-cell large granular lymphocyte proliferations with subsequent progression to systemic TCL. The third theme included 6 patients with TCL with T-follicular helper phenotype, mainly angioimmunoblastic T-cell lymphoma, with concurrent/subsequent clonal hematopoiesis or myeloid neoplasms and/or subsequent/concomitant diffuse large B-cell lymphoma. CONCLUSIONS: This cohort of cases allowed us to illustrate, discuss, and review current concepts of transdifferentiation, aberrant antigen expression, and progression in various T/NK-cell neoplasms.

Progression of follicular lymphoma and related entities: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: The 2021 Society for Hematopathology and European Association for Haematopathology Workshop addressed the molecular and cytogenetic underpinnings of transformation and transdifferentiation in lymphoid neoplasms. METHODS: Session 4, "Transformations of Follicular Lymphoma," and session 5, "Transformations of Other B-Cell Lymphomas," included 45 cases. Gene alteration analysis and expression profiling were performed on cases with submitted formalin-fixed, paraffin embedded tissue. RESULTS: The findings from session 4 suggest that "diffuse large B-cell lymphoma/high-grade B-cell lymphoma with rearrangements of MYC and BCL2" is a distinct category arising from the constraints of a preexisting BCL2 translocation. TdT expression in aggressive B-cell lymphomas is associated with MYC rearrangements, immunophenotypic immaturity, and a dismal prognosis but must be differentiated from lymphoblastic -lymphoma. Cases in session 5 illustrated unusual morphologic and immunophenotypic patterns of transformation. Additionally, the findings support the role of cytogenetic abnormalities-specifically, MYC and NOTCH1 rearrangements-as well as single gene alterations, including TP53, in transformation. CONCLUSIONS: Together, these unique cases and their accompanying molecular and cytogenetic data suggest potential mechanisms for and unusual patterns of transformation in B-cell lymphomas and indicate numerous opportunities for further study.

Transformations of marginal zone lymphomas and lymphoplasmacytic lymphomas: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: To summarize the conclusions of the 2021 Society for Hematopathology/European Association for Haematopathology workshop regarding transformations of marginal zone lymphoma (MZL) and lymphoplasmacytic lymphoma (LPL). METHODS: Nineteen cases were submitted to this portion of the workshop. Additional studies were performed in cases with sufficient material. RESULTS: Cases included splenic MZL (n = 4), splenic diffuse red pulp small B-cell lymphoma (n = 2), nodal MZL (n = 4), extranodal MZL (n = 1), and LPL (n = 8). The most common transformation was to diffuse large B-cell lymphoma (DLBCL), but others included classic Hodgkin lymphoma, high-grade B-cell lymphomas with MYC and BCL6 rearrangements, plasmablastic lymphoma, and plasma cell leukemia. Two splenic MZLs with transformation to DLBCL contained t(14;19)(q32;q13.3) IGH::BCL3 rearrangements in both samples. Paired sequencing studies in 5 MZLs with transformation to clonally related DLBCL identified a variety of mutations and gene fusions at the time of transformation, including CARD11, IGH::MYC, NOTCH2, P2RY8, TBLX1X1, and IGH::CD274. CONCLUSIONS: Marginal zone lymphoma and LPL may undergo a variety of transformation events, most commonly to DLBCL, which is usually, although not always, directly clonally related to the underlying low-grade lymphoma. Multiparameter analysis including broad-based sequencing studies can assist in the diagnosis and classification of these uncommon cases.

Progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma and B-cell prolymphocytic leukemia: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: Session 3 of the 2021 Workshop of the Society for Hematopathology/European Association for Haematopathology examined progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and B-cell prolymphocytic leukemia (B-PLL). METHODS: Thirty-one cases were reviewed by the panel. Additional studies such as immunohistochemistry and molecular genetic testing, including whole-exome sequencing and expression profiling, were performed in select cases. RESULTS: Session 3 included 27 CLL/SLL cases and miscellaneous associated proliferations, 3 cases of B-PLL, and 1 case of small B-cell lymphoma. The criteria for -accelerated CLL/SLL are established for lymph nodes, but extranodal disease can be diagnostically challenging. Richter transformation (RT) is a broad term and includes true transformation from original CLL/SLL clone(s) and clonally unrelated neoplasms. The morphologic, immunophenotypic, and genetic spectrum is diverse with classical and highly unusual examples. T-cell proliferations can also be encountered in CLL/SLL. B-cell prolymphocytic leukemia is a rare, diagnostically challenging disease due to its overlaps with other lymphoid neoplasms. CONCLUSIONS: The workshop highlighted complexity of progression and transformation in CLL/SLL and B-PLL, as well as diagnostic caveats accompanying heterogeneous presentations of RT and other manifestations of disease progression. Molecular genetic studies are pivotal for diagnosis and determination of clonal relationship, and to predict response to treatment and identify resistance to targeted therapy.

B-cell lineage neoplasms transdifferentiating into histiocytic/dendritic cell neoplasms: diversity, differentiation lineage, genomic alterations, and therapy: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: To report findings from the 2021 Society for Hematopathology/European Association for Haematopathology Workshop within the category of B-cell lineage neoplasms' transdifferentiation into histiocytic/dendritic cell neoplasms (HDCNs). METHODS: The workshop panel reviewed 29 cases, assigned consensus diagnoses, and summarized findings. RESULTS: The specific diagnoses of transdifferentiated HDCN tumors were histiocytic sarcoma (16); Langerhans cell histiocytosis/sarcoma (5); indeterminate DC tumor (1); and HDCN, unclassifiable (1). Approximately one-third of the patients reviewed had follicular lymphoma; lymphoblastic leukemia/lymphoma; or another B-cell lymphoma, most commonly chronic lymphocytic leukemia/small lymphocytic lymphoma. There was a 3:1 preponderance toward women, median patient age was 60 years, and the median interval between the initial diagnosis of B-cell lineage neoplasm and HDCN was 4 to 5 years. The submitted cases have demonstrated substantial heterogeneity as well as overlapping immunophenotypic and other features. Comprehensive genomic DNA sequencing revealed alterations enriched in the MAPK pathway. Based on shared and distinct alterations seen in HDCNs and the preceding lymphomas, both linear and divergent clonal evolutionary pathways were inferred. Furthermore, RNA sequencing performed in a subset of cases yielded new insights into markers that could be useful for more precise cell lineage identification. The panel has thus proposed an updated algorithm for HDCN lineage assignment. The outcome of transdifferentiated HDCNs was poor, but the MAPK signaling pathway emerges as a potentially attractive therapeutic target. CONCLUSIONS: Transdifferentiated HDCNs demonstrate heterogeneity and pose diagnostic challenges with regard to exact classification, but the in-depth characterization of the submitted cases have added to our understanding of the secondary HDCNs transdifferentiated from B-cell lymphoma/leukemia. Continuous efforts focusing on deciphering the specific cell lineage and differentiation state of these tumors will be critical for their accurate classification. Comprehensive molecular characterization of HDCNs may be informative in this regard. With the list of novel pharmacologic inhibitors of the MAPK pathway continuing to expand, improved outcomes for HDCN can be expected.

Phenotypic and genotypic infidelity in B-lineage neoplasms, including transdifferentiation following targeted therapy: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: Session 2 of the 2021 Society for Hematopathology and European Association for Haematopathology Workshop collected examples of lineage infidelity and transdifferentiation in B-lineage neoplasms, including after targeted therapy. METHODS: Twenty cases were submitted. Whole-exome sequencing and genome-wide RNA expression analysis were available on a limited subsample. RESULTS: A diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) was rendered on at least 1 biopsy from 13 patients. There was 1 case of acute myeloid leukemia (AML); the remaining 6 cases were mature B-cell neoplasms. Targeted therapy was administered in 7 cases of B-ALL and 4 cases of mature B-cell neoplasms. Six cases of B-ALL underwent lineage switch to AML or mixed-phenotype acute leukemia at relapse, 5 of which had rearranged KMT2A. Changes in maturational state without lineage switch were observed in 2 cases. Examples of de novo aberrant T-cell antigen expression (n = 2) were seen among the mature B-cell lymphoma cohort, and their presence correlated with alterations in tumor cell gene expression patterns. CONCLUSIONS: This cohort of cases enabled us to illustrate, discuss, and review current concepts of lineage switch and aberrant antigen expression in a variety of B-cell neoplasms and draw attention to the role targeted therapies may have in predisposing neoplasms to transdifferentiation as well as other, less expected changes in maturational status.

Progression of Hodgkin lymphoma and plasma cell neoplasms: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: To summarize cases submitted to the 2021 Society for Hematopathology/European Association for Haematopathology Workshop under the categories of progression of Hodgkin lymphoma, plasmablastic myeloma, and plasma cell myeloma. METHODS: The workshop panel reviewed 20 cases covered in this session. In addition, whole-exome sequencing (WES) and whole-genome RNA expression analysis were performed on 10 submitted cases, including 6 Hodgkin lymphoma and 4 plasma neoplasm cases. RESULTS: The cases of Hodgkin lymphoma included transformed cases to or from various types of B-cell lymphoma with 1 exception, which had T-cell differentiation. The cases of plasma cell neoplasms included cases with plasmablastic progression, progression to plasma cell leukemia, and secondary B-lymphoblastic leukemia. Gene variants identified by WES included some known to be recurrent in Hodgkin lymphoma and plasma cell neoplasm. All submitted Hodgkin lymphoma samples showed 1 or more of these mutations: SOCS1, FGFR2, KMT2D, RIT1, SPEN, STAT6, TET2, TNFAIP3, and ZNF217. CONCLUSIONS: Better molecular characterization of both of these neoplasms and mechanisms of progression will help us to better understand mechanisms of progression and perhaps develop better prognostic models, as well as identifying novel therapeutic targets.

Bonded cumomer analysis of tumor metabolism based on 13 C magnetic resonance spectroscopy.

Bonded cumomers are sets of isotopomers of 13 C-labeled metabolites containing a particular sequence of contiguously or singly labeled carbon atoms. Only these isotopomers contribute to multiplet structure in the 13 C NMR spectrum. We discuss the application of this technique to the study of quantitative tumor metabolism, bioenergetics, and the Warburg effect. The advantages and sensitivity of bonded cumomer analysis over positional enrichment analysis are discussed. When sensitivity requirements are met, bonded cumomer analysis enables the extraction of fluxes through specific metabolic pathways with higher precision. In conjunction with isotopomer control analysis, we evaluate the sensitivity of experimentally measurable metabolite multiplets to determine the robustness of flux analysis in 13 C spectra of tumors. This review examines the role of glycolytic and tricarboxylic acid cycle metabolism with special emphasis on flux through the pentose phosphate pathway (PPP). The impact of reversibility of the nonoxidative branch of the PPP with various 13 C glucose tracers on fine-structure multiplets is analyzed.

DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.

Cell block-based RNA next generation sequencing for detection of gene fusions in lung adenocarcinoma: An institutional experience.

OBJECTIVE: Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test. METHODS: Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS. RESULTS: A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material. CONCLUSIONS: Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.

Induction of Transcriptional Inhibitor HES1 and the Related Repression of Tumor-Suppressor TXNIP Are Important Components of Cell-Transformation Program Imposed by Oncogenic Kinase NPM-ALK.

This study reports that hairy and enhancer of split homolog-1 (HES1), known to repress gene transcription in progenitor cells of several cell lineages, was strongly expressed in cells and tissues of T-cell lymphoma expressing the oncogenic chimeric tyrosine kinase nucleophosmin (NPM)-anaplastic lymphoma kinase [ALK; ALK+ T-cell lymphoma (TCL)]. The structural analysis of the Orange domain of HES1 indicated that HES1 formed a highly stable homodimer. Of note, repression of HES1 expression led to inhibition of ALK+ TCL cell growth in vivo. The expression of the HES1 gene was induced by NPM-ALK through activation of STAT3, which bound to the gene's promoter and induced the gene's transcription. NPM-ALK also directly phosphorylated HES1 protein. In turn, HES1 up-regulated and down-regulated in ALK+ TCL cells, the expression of numerous genes, protein products of which are involved in key cell functions, such as cell proliferation and viability. Among the genes inhibited by HES1 was thioredoxin-interacting protein (TXNIP), encoding a protein implicated in promotion of cell death in various types of cells. Accordingly, ALK+ TCL cells and tissues lacked expression of TXNIP, and its transcription was co-inhibited by HES1 and STAT3 in an NPM-ALK-dependent manner. Finally, the induced expression of TXNIP induced massive apoptotic cell death of ALK+ TCL cells. The results reveal a novel NPM-ALK-controlled pro-oncogenic regulatory network and document an important role of HES and TXNIP in the NPM-ALK-driven oncogenesis, with the former protein displaying oncogenic and the latter tumor suppressor properties.

NPM-ALK-Induced Reprogramming of Mature TCR-Stimulated T Cells Results in Dedifferentiation and Malignant Transformation.

Fusion genes including NPM-ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM-ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM-ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)-generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM-ALK translocations. These findings describe the fundamental mechanisms of NPM-ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. SIGNIFICANCE: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM-ALK, and delineates dynamic transcriptional changes as a T cell transforms.See related commentary by Spasevska and Myklebust, p. 3160.

Ibrutinib and venetoclax target distinct subpopulations of CLL cells: implication for residual disease eradication.

Ibrutinib inhibits Bruton tyrosine kinase while venetoclax is a specific inhibitor of the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL), and clinical trials using the combination therapy have produced remarkable results in terms of rate of complete remission and frequency of undetectable minimal residual disease. However, the laboratory rationale behind the success of the drug combination is still lacking. A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. Using an ex vivo model that promotes CLL proliferation, we show that modeled ibrutinib proliferative responses, but not viability responses, correlate well with patients' actual clinical responses. Importantly, we demonstrate for the first time that ibrutinib and venetoclax act on distinct CLL subpopulations that have different proliferative capacities. While the dividing subpopulation of CLL responds to ibrutinib, the resting subpopulation preferentially responds to venetoclax. The combination of these targeted therapies effectively reduced both the resting and dividing subpopulations in most cases. Our laboratory findings help explain several clinical observations and contribute to the understanding of tumor dynamics. Additionally, our proliferation model may be used to identify novel drug combinations with the potential of eradicating residual disease.