Transgenic Hematopoietic Stem Cells Research Articles

Abstract 1771: Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma

Abstract Background: Front-line chemoimmunotherapy for diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is effective, however at least 40% of patients relapse or are refractory (r/r). Of this r/r DLBCL cohort, approximately 10% will be cured with conventional second-line treatments consisting of salvage chemotherapy and autologous stem cell transplant. CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new second-line options in diffuse large B-cell lymphoma (DLBCL) and show exciting 30-40% durable complete responses in heavily pretreated patients. Recent whole-genome sequencing data published by our lab revealed that deletion of Ras homolog family member A (RHOA) is strongly associated with poor response to CAR-19 therapy in r/r DLBCL patients (p = 0.0013). In newly diagnosed DLBCL, 20% of patients harbor a RHOA deletion and another 5% have loss-of-function mutations. To date, no work has comprehensively identified the role of RHOA in DLBCL or how its inactivation may protect CAR-T resistant tumors. Methods: We created a novel in vivo model of RHOA deficient lymphoma by transplanting Eμ-Myc transgenic hematopoietic stem cells (HSCs) transduced with a shRhoa vector. We also created a syngeneic model of RHOA-deficient lymphoma with MYC-driven BL3750 cells. In vitro flow cytometry and Xcelligence real-time cell analysis experiments were utilized to assess cytotoxicity of CAR-19 against RHOA-deficient lymphoma cell lines. RNAseq analysis was conducted on Rhoa knockdown cell lines to elucidate cell-intrinsic mechanisms of lymphomagenesis. Cell proliferation and migration assays were performed to characterize the phenotype of RHOA-deficient DLBCL cell lines. Results: 18 mice were transplanted (11 shRhoa, 7 shRenilla control) and are being monitored for lymphoma development. In vitro CAR-T experiments demonstrated that while RHOA-deficient cells demonstrate a similar degree of killing initially, a population of RHOA-deficient cells persists and enrich at later time points. RNA-seq analysis of A20 shRhoa murine lymphoma cells uncovered enrichment of gene sets associated will cell-cycle progression and interferon (INF) signaling pathways. Cell proliferation assay and Western blot of RHOA inactivated cell lines revealed increased proliferation in shRhoa cells through the AKT/mTOR pathway compared to controls. Conclusions: RHOA-deficient lymphomas, while not entirely evading CAR-19 cells in vitro, demonstrate resistance mechanisms that allows a small population to persist. RNA-seq analysis reveals upregulated oncogenic and immunoinhibitory pathways, consistent with in vitro cell proliferation and Western blot assays. Further research will challenge in vivo RHOA-deficient tumors with CAR-19 cells and employ scRNA-seq to reveal transcriptomic changes within RHOA-deficient lymphoma responsible for CAR-19 resistance. Citation Format: Austin Newsam, Yitzhar Goretsky, Evan Roberts, Daniel Bilbao, Jonathan Schatz. Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1771.

DLBCL-Derived NF-κb Mutations Exploit Senescence and Immune Evasion Mechanisms to Promote Myc-Driven Lymphomagenesis

Introduction: Constitutive NF-κB activation is a hallmark feature of B-cell malignancies, including the ABC-subtype of diffuse large B-cell lymphoma (DLBCL), and is mainly due to gain-of-function mutations in components of the Toll-like receptor (MYD88) or B-cell receptor (CD79B, CARD11) pathways, inactivating mutations of cellular NF-κB suppressors (TNFAIP3/A20, BIRC3) or copy number gain (NFKBIZ), often in the presence of high Myc oncoprotein expression. In order to understand whether such genetic lesions may cooperate with Myc during lymphomagenesis, we took advantage of the Eµ- myc transgenic mouse in which aggressive B-cell lymphomas are induced by B-cell-specific overexpression of c-Myc.Methods: Eµ- myc transgenic hematopoietic stem cells (HSC) were stably transduced with GFP-expressing retroviral vectors encoding the murine orthologs of human MYD88-L265P, NFKBIZ, CD79B-ITAM-Y196H, CARD11-L244P, CARD11-L225LI or shRNA against TNFAIP3/A20 or BIRC3 prior to their transplantation into lethally irradiated syngenic C57BL/6 mice. Recipient animals were subsequently monitored for the development of a palpable lymphadenopathy, and lymphomas were subject to immunophenotyping, analysis of growth parameters and transcriptome profiling by RNA-Seq.Results : Ectopic overexpression of NFKBIZ, MYD88-L265P, CD79B-ITAM-Y196H or knockdown of TNFAIP3/A20 in Eµ- myc transgenic HSC gave rise to GFP(+) B-cell lymphomas (> 70% GFP+/B220+ cells), albeit at varying frequencies. All mutant-driven lymphomas manifested with reduced apoptotic TUNEL/B220 indices when compared to the empty-vector control cohort (ctrl. lymphomas). In contrast, neither knockdown of BIRC3 nor overexpression of the CARD11 mutants generated GFP(+) Eµ- myc lymphomas in vivo . Unexpectedly, we found senescent GFP(+) B- lymphocytes residing in the spleens of these healthy animals after transplantation. Genetic ablation of the senescence program (via allelic deletion of the histone demethylase Suv39h1 or the INK4a/ARF locus) in Eµ- myc HSC prior to transduction with the CARD11 mutants gave rise to palpable GFP(+) B-cell lymphomas (unlike the empty-vector), indicating that CARD11 missense mutations require a senescence-deficient background to promote Eµ- myc -driven lymphomagenesis.RNA-Seq unvealed downregulation of MHC class II genes as a common feature of all mutant-driven Eµ- myc lymphomas when compared to ctrl. lymphomas. Since MHC/T-cell receptor interactions are required for an effective anti-tumoral adaptive immune response, this finding suggested immune evasion mechanisms to be exploited by all genetic lesions tested to promote Myc-driven lymphomagenesis. Further supporting this notion, MYD88-L265P-driven lymphomas exhibited elevated surface expression of the immune checkpoint ligands PD-L1 and PD-L2. Strikingly, knockdown of PD-L1 alone was sufficient to significantly delay MYD88-L265P-driven lymphomagenesis in Eµ- myc HSC transplantation experiments, suggesting that PD-L1-mediated immune evasion mechanisms contribute significantly to the aggressive phenotype observed under the MyD88-L265P mutant in vivo . Furthermore, overexpression of PD-L1 in Eµ- myc HSC alone also induced GFP(+) B-cell lymphomas that displayed a striking overlap in gene expression with MyD88-L265P-driven Eµ- myc lymphomas.Conclusions: We continue to characterize the Eµ- myc transgenic mouse as a well-suited model platform to functionally dissect DLBCL-relevant oncogenic networks in general and NF-κB-activating mutations in the context of high Myc protein expression, a frequent feature of DLBCL, in particular. Our systematic in vivo analysis of different NF-κB-activating mutants - so far rather recognized as functionally interchangeable moieties - indicated that some of these genetic lesions directly cooperate with Myc (i.e. MyD88-L265P, NFKBIZ) - partly via utilizing immune evasion mechanisms - while others (e.g. CARD11 mutations) require upfront senescence-ablating lesions to license Myc-driven lymphomagenesis. Our findings might explain the pressure to select for senescence-ablating lesions like INK4a/ARF loss frequently found in human DLBCL, and could inform immune checkpoint blockade treatment strategies based on certain oncogenic/immune evasion network interconnections. DisclosuresNo relevant conflicts of interest to declare.

Adaptive T-cell immunity controls senescence-prone MyD88- or CARD11-mutant B-cell lymphomas

AbstractAberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor β (TGF-β)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti–programmed cell death 1 checkpoint blockade, leading to direct T-cell–mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.

Modeling the CNS Tropism of Diffuse Large B-Cell Lymphomas in Vivo

Introduction: Central nervous system (CNS) manifestation accounts for dismal prognosis in patients diagnosed with systemic diffuse large B-cell lymphoma (DLBCL). Myc rearrangement, deletions encompassing the CDKN2A (a.k.a. INK4a/ARF) or ATM gene loci as well as NF-kB-hyperactivating mutations are frequently detected in primary and secondary CNS-tropic lymphoma, however, investigations that functionally link these lesions to CNS involvement in adequate in vivo model systems are missing.Methods: We generated primary Eµ-myc transgenic mouse lymphomas with and without distinct naturally occurring NF-kB mutations (within genes encoding for MyD88, CD79B, A20, IkBζ, IkBε or BIRC3) or deletions at the INK4a/ARF and ATM loci by retroviral gene transfer and crossbreeding to the respective knockout strains. A subset of the lymphomas was subjected to gene expression profiling and whole-exome sequencing (WES). Wild-type recipient mice propagated with lymphoma cells via tail vein injection were monitored for systemic lymphoma development, the time at which the brain was isolated and examined regarding lymphoma infiltration.Results: Underlining Myc's role as a putative co-driver of CNS involvement, we found in about 40% of primary Eµ-myc lymphomas (with no additional exogenous lesions) meningeal lymphoma manifestations, and transplantation of the same individual lymphomas into numerous recipients reproduced the CNS lymphoma status. Gene set enrichment analysis of genome-wide transcriptome profiles indicated NF-kB hyperactivation in the CNS-tropic lymphoma group, suggesting that constitutive NF-kB signaling may promote CNS-prone pathogenesis in vivo. Transplantation of Eµ-myc transgenic hematopoietic stem cells expressing a variety of NF-kB-activating mutants in myeloablated recipient mice resulted in a significant acceleration of Eµ-myc -driven lymphomagenesis, with some, but not all of these mutants conferring a CNS-tropic lymphoma phenotype. Global NF-kB suppression in CNS-tropic Eµ-myc lymphomas via the NF-kB-antagonizing IkBΔN super-repressor did not fully abrogate lymphoma infiltration of the brain, suggesting that additional factor(s) must contribute. Accordingly, targeted ablation of the INK4a/ARF and ATM loci robustly enhanced CNS tropism of Eµ-myc lymphomas.Conclusions: The Eµ-myc mouse lymphoma model is well-suited to genetically dissect and rebuild components of DLBCL-like CNS tropism. We identified CDKN2A or ATM deletions as critical determinants of CNS tropism in vivo. Our systematic analyses of different NF-kB mutants - so far rather recognized as functionally interchangeable - indicated that only distinct NF-kB mutants contribute to CNS tropism in B-cell lymphomas. WES data, results from compound genotypes (e.g. combining ATM deletions with an NF-kB-activating mutation), and the impact of the host's cellular immune status will be reported at the meeting. Our findings underscore the need for functional analyses of oncogenic network contexts, and provide important insights into candidate target lesions for personalized CNS-directed therapies in DLBCL patients in the future. DisclosuresNo relevant conflicts of interest to declare.

FoxO transcription factors suppress Myc-driven lymphomagenesis via direct activation of Arf

FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Here we show that stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Emu-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Emu-myc; p53(+/-) progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19(Arf) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. We find that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity.

Overexpression of a hematopoietic transcriptional regulator EDAG induces myelopoiesis and suppresses lymphopoiesis in transgenic mice

Erythroid differentiation-associated gene (EDAG) is a hematopoietic tissue-specific gene that is highly expressed in the earliest CD34+ lin- bone marrow (BM) cells and involved in the proliferation and differentiation of hematopoietic cells. To investigate the role of EDAG in hematopoiesis, we established an EDAG transgenic mouse model driven by human CD11a promoter. The transgenic mice showed increased mortality with severe organ infiltration by neutrophils, and the homeostasis of hematopoiesis was broken. The myelopoiesis was enhanced with expansion of myeloid cells in BM, increased peripheral granulocytes and extramedullary myelopoiesis in spleen. In contrast to myeloid cells, the lymphoid commitment was severely impaired with the B lymphopoiesis blocked at the transition from pro/pre-B I to pre-B II stage in BM and T thymocytes development blocked at the most immature stage (DN I). Moreover, we showed that EDAG was a transcriptional regulator which had transactivation activity and regulated the expression of several key transcription factors such as PU.1 and Pax5 in transgenic hematopoietic stem cells. These data suggested that EDAG was a key transcriptional regulator in maintaining the homeostasis of hematopoietic lineage commitment.

Simple and Efficient Isolation of Hematopoietic Stem Cells from H2K-zFP Transgenic Mice

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

Donor Marker Infidelity in Transgenic Hematopoietic Stem Cells

Transgenic marking approaches are increasingly used to evaluate the developmental potential of stem cells. However, cell fate mapping studies using different transgenic marking systems have produced conflicting results. These disparate findings may be due in part to the infidelity of donor marker gene expression. Analysis of hematopoietic stem cells (c-Kit+, Sca-1+, lineage marker- [KSL]) from a transgenic mouse (1Osb) engineered to ubiquitously express the enhanced green fluorescent protein (EGFP) reveals two distinct populations. Forty percent of KSL cells demonstrate intermediate levels of EGFP fluorescence and differentiate into subpopulations of B cells, T cells, and myeloid cells that do not express EGFP. By contrast, progeny of the remaining 60% of KSL cells are almost exclusively EGFP bright. Long-term multilineage hematopoietic reconstitution and serial transplantation experiments show that these differences in EGFP are a property of self-renewing stem cells. Furthermore, both the transgene integration site and the activation status of a cell are important determinants of EGFP expression. These results indicate that a combination of donor cell markers is required to reliably track the full differentiation potential of transgenic stem cells.

In vivo selection of antifolate-resistant transgenic hematopoietic stem cells in a murine bone marrow transplant model.

Currently, low levels of stable gene transfer into hematopoietic tissues of large animals and humans continues to limit the clinical application of gene therapy. One strategy for overcoming this problem is to selectively expand, in vivo, the population of successfully gene-modified cells. Recent work has shown that nucleoside transport inhibition in combination with antifolates can be used to select in vivo for hematopoietic stem cells expressing drug-resistant dihydrofolate reductase (DHFR). In this study we investigated whether trimetrexate (TMTX) and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose phosphate (NBMPR-P) can be used to select for tyr22-variant DHFR expressing transgenic hematopoietic cells in a murine bone marrow transplant model. Our results indicate that 40 mg/kg TMTX and 20 mg/kg NBMPR-P can be used in combination to expand transgene-positive progenitor cells 3- to 4-fold immediately following drug administration. In addition, long-term progenitor populations were expanded 2- to 3-fold in primary recipients, to approximately 5 months following drug administration. Secondary transplants conducted with marrow from primary recipients 5 months following drug administration revealed a statistically significant selective expansion of transgene-positive cells in the spleens and peripheral blood of these animals. No such expansion was observed in groups of mice treated with TMTX alone or NBMPR-P alone. We conclude that TMTX + NBMPR-P can be used to selectively expand transgenic tyr22-variant DHFR expressing murine hematopoietic stem cells in vivo.