EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma.

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

Effective treatment with the selective cytokine inhibitor BNZ-1 reveals the cytokine dependency of T-LGL leukemia.

T-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes that can result in severe neutropenia, anemia, and bone marrow failure. Strong evidence from patients and mouse models demonstrate the critical role of interleukin-15 (IL-15) in T-LGLL pathogenesis. BNZ-1 is a pegylated peptide that selectively inhibits the binding of IL-15 and other γc cytokines to their cellular receptor complex, which has demonstrated efficacy in ex vivo T-LGLL cells and transgenic mice in preclinical studies. We conducted a phase 1/2 trial of BNZ-1 in patients with T-LGLL who had hematocytopenias (anemia or neutropenia) and required therapy. Clinical responses were assessed using hematologic parameters (improvement in hematocytopenias) based on response criteria from the Eastern Cooperative Oncology Group 5998 T-LGLL trial. BNZ-1 demonstrated clinical partial responses in 20% of patients with T-LGLL with minimal toxicity and the maximum tolerated dose was not reached. Furthermore, T-LGL leukemic cells showed significantly increased apoptosis in response to BNZ-1 treatment as early as day 2, including in clinical nonresponders, with changes that remained statistically different from baseline throughout treatment (P < .005). We report first-in-human proof that T-LGL leukemic cells are dependent on IL-15 and that intervention with IL-15 inhibition with BNZ-1 in patients with T-LGLL shows therapeutic effects, which carries important implications for the understanding of the pathogenesis of this disease. This trial was registered at www.clinicaltrials.gov as #NCT03239392.

Analysis and therapeutic targeting of the IL-1R pathway in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), a subgroup of mature T-cell neoplasms with an aggressive clinical course, is characterized by elevated expression of CD30 and anaplastic cytology. To achieve a comprehensive understanding of the molecular characteristics of ALCL pathology and to identify therapeutic vulnerabilities, we applied genome-wide CRISPR library screenings to both anaplastic lymphoma kinase positive (ALK+) and primary cutaneous (pC) ALK- ALCLs and identified an unexpected role of the interleukin-1R (IL-1R) inflammatory pathway in supporting the viability of pC ALK- ALCL. Importantly, this pathway is activated by IL-1α in an autocrine manner, which is essential for the induction and maintenance of protumorigenic inflammatory responses in pC-ALCL cell lines and primary cases. Hyperactivation of the IL-1R pathway is promoted by the A20 loss-of-function mutation in the pC-ALCL lines we analyze and is regulated by the nonproteolytic protein ubiquitination network. Furthermore, the IL-1R pathway promotes JAK-STAT3 signaling activation in ALCLs lacking STAT3 gain-of-function mutation or ALK translocation and enhances the sensitivity of JAK inhibitors in these tumors in vitro and in vivo. Finally, the JAK2/IRAK1 dual inhibitor, pacritinib, exhibited strong activities against pC ALK- ALCL, where the IL-1R pathway is hyperactivated in the cell line and xenograft mouse model. Thus, our studies revealed critical insights into the essential roles of the IL-1R pathway in pC-ALCL and provided opportunities for developing novel therapeutic strategies.

Accessory cells precondition naïve T cells and regulatory T cells for cytokine-mediated proliferation.

Naïve T cells and regulatory T cells, when purified, do not proliferate to the γc-cytokines IL-2, IL-7, or IL-15, despite their expression of cognate cytokine receptors. Dendritic cells (DCs) enabled the T cell proliferation to these cytokines, through cell-to-cell contact, but independent of T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in DC-depleted hosts. We propose calling this a "preconditioning effect". Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2 target genes. "Preconditioning" was necessary to activate these two pathways and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation, and prolonged S6 phosphorylation occurred. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.

Interleukin-15 augments NK cell-mediated ADCC of alemtuzumab in patients with CD52+ T-cell malignancies.

Interleukin-15 (IL-15) monotherapy substantially increases the number and activity of natural killer (NK) cells and CD8+ T cells but has not produced clinical responses. In a xenograft mouse model, IL-15 enhanced the NK cell-mediated antibody-dependent cell cytotoxicity (ADCC) of the anti-CD52 antibody alemtuzumab and led to significantly more durable responses than alemtuzumab alone. To evaluate whether IL-15 potentiates ADCC in humans, we conducted a phase 1 single-center study of recombinant human IL-15 and alemtuzumab in patients with CD52-positive mature T-cell malignances. We gave IL-15 subcutaneously 5 days per week for 2 weeks in a 3 + 3 dose escalation scheme (at 0.5, 1, and 2 µg/kg), followed by standard 3 times weekly alemtuzumab IV for 4 weeks. There were no dose-limiting toxicities or severe adverse events attributable to IL-15 in the 11 patients treated. The most common adverse events were lymphopenia (100%), alemtuzumab-related infusion reactions (90%), anemia (90%), and neutropenia (72%). There were 3 partial and 2 complete responses, with an overall response rate of 45% and median duration of response 6 months. Immediately after 10 days of IL-15, there was a median 7.2-fold increase in NK cells and 2.5-fold increase in circulating CD8+ T cells, whereas the number of circulating leukemic cells decreased by a median 38% across all dose levels. Treatment with IL-15 was associated with increased expression of NKp46 and NKG2D, markers of NK-cell activation, and increased ex vivo ADCC activity of NK cells, whereas inhibitory receptors PD1 and Tim3 were decreased. This trial was registered at www.clinicaltrials.gov as #NCT02689453.

The implications of IL-15 trans-presentation on the immune response.

Interleukin-15 is a pleiotropic cytokine type I four alpha-helical bundle cytokine that along with IL-2, IL-4, IL-7, IL-9, and IL-21 shares the common cytokine receptor γ chain, γc. IL-15 is vital for the development, survival, and expansion of natural killer cells and for the development of CD8+ memory T cells. Whereas other family γc cytokines signal by directly binding to their target cells, IL-15 is distinctive in that it binds to IL-15Rα, a sushi domain containing binding protein that is expressed on a number of cell types, including monocytes and dendritic cells as well as T cells, and then is trans-presented to responding cells that express IL-2Rß and γc. This distinctive mechanism for IL-15 relates to its role in signaling in the context of cell-cell interactions and signaling synapses. The actions of IL-15 and ways of manipulating its actions to potential therapeutic benefit are discussed.

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.

IL15 and Anti-PD-1 Augment the Efficacy of Agonistic Intratumoral Anti-CD40 in a Mouse Model with Multiple TRAMP-C2 Tumors.

PURPOSE: IL15 promotes activation and maintenance of natural killer (NK) and CD8+ T effector memory cells making it a potential immunotherapeutic agent for the treatment of cancer. However, monotherapy with IL15 was ineffective in patients with cancer, indicating that it would have to be used in combination with other anticancer agents. The administration of high doses of common gamma chain cytokines, such as IL15, is associated with the generation of "helpless" antigen-nonspecific CD8 T cells. The generation of the tumor-specific cytotoxic T cells can be mediated by CD40 signaling via agonistic anti-CD40 antibodies. Nevertheless, parenteral administration of anti-CD40 antibodies is associated with unacceptable side effects, such as thrombocytopenia and hepatic toxicity, which can be avoided by intratumoral administration. EXPERIMENTAL DESIGN: We investigated the combination of IL15 with an intratumoral anti-CD40 monoclonal antibody (mAb) in a dual tumor TRAMP-C2 murine prostate cancer model and expanded the regimen to include an anti-PD-1 mAb. RESULTS: Here we demonstrated that anti-CD40 given intratumorally not only showed significant antitumor activity in treated tumors, but also noninjected contralateral tumors, indicative of abscopal efficacy. The combination of IL15 with intratumoral anti-CD40 showed an additive immune response with an increase in the number of tumor-specific tetramer-positive CD8 T cells. Furthermore, the addition of anti-PD-1 further improved efficacy mediated by the anti-CD40/IL15 combination. CONCLUSIONS: These studies support the initiation of a clinical trial in patients with cancer using IL15 in association with the checkpoint inhibitor, anti-PD-1, and intratumoral optimized anti-CD40.

A very long-acting IL-15: implications for the immunotherapy of cancer.

BACKGROUND: Interleukin-15 (IL-15) is an important cytokine necessary for proliferation and maintenance of natural killer (NK) and CD8+ T cells, and with great promise as an immuno-oncology therapeutic. However, IL-15 has a very short half-life and a single administration does not provide the sustained exposure required for optimal stimulation of target immune cells. The purpose of this work was to develop a very long-acting prodrug that would maintain IL-15 within a narrow therapeutic window for long periods-similar to a continuous infusion. METHODS: We prepared and characterized hydrogel microspheres (MS) covalently attached to IL-15 (MS~IL-15) by a releasable linker. The pharmacokinetics and pharmacodynamics of MS~IL-15 were determined in C57BL/6J mice. The antitumor activity of MS~IL-15 as a single agent, and in combination with a suitable therapeutic antibody, was tested in a CD8+ T cell-driven bilateral transgenic adenocarcinoma mouse prostate (TRAMP)-C2 model of prostatic cancer and a NK cell-driven mouse xenograft model of human ATL (MET-1) murine model of adult T-cell leukemia. RESULTS: On subcutaneous administration to mice, the cytokine released from the depot maintained a long half-life of about 168 hours over the first 5 days, followed by an abrupt decrease to about ~30 hours in accordance with the development of a cytokine sink. A single injection of MS~IL-15 caused remarkably prolonged expansions of NK and ɣδ T cells for 2 weeks, and CD44hiCD8+ T cells for 4 weeks. In the NK cell-driven MET-1 murine model of adult T-cell leukemia, single-agent MS~IL-1550 µg or anti-CCR4 provided modest increases in survival, but a combination-through antibody-depedent cellular cytotoxicity (ADCC)-significantly extended survival. In a CD8+ T cell-driven bilateral TRAMP-C2 model of prostatic cancer, single agent subcutaneous MS~IL-15 or unilateral intratumoral agonistic anti-CD40 showed modest growth inhibition, but the combination exhibited potent, prolonged bilateral antitumor activity. CONCLUSIONS: Our results show MS~IL-15 provides a very long-acting IL-15 with low Cmax that elicits prolonged expansion of target immune cells and high anticancer activity, especially when administered in combination with a suitable immuno-oncology agent.

Triple combination of BET plus PI3K and NF-κB inhibitors exhibit synergistic activity in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

Phase I study of single agent NIZ985, a recombinant heterodimeric IL-15 agonist, in adult patients with metastatic or unresectable solid tumors.

BACKGROUND: NIZ985 is a recombinant heterodimer of physiologically active interleukin (IL-)15 and IL-15 receptor alpha. In preclinical models, NIZ985 promotes cytotoxic lymphocyte proliferation, killing function, and organ/tumor infiltration, with resultant anticancer effects. In this first-in-human study, we assessed the safety, pharmacokinetics, and immune effects of NIZ985 in patients with metastatic or unresectable solid tumors. METHODS: Single agent NIZ985 dose escalation data are reported from a phase I dose escalation/expansion study of NIZ985 as monotherapy. Adult patients (N=14) received 0.25, 0.5, 1, 2 or 4 µg/kg subcutaneous NIZ985 three times weekly (TIW) for the first 2 weeks of each 28-day cycle, in an accelerated 3+3 dose escalation trial design. IL-15 and endogenous cytokines were monitored by ELISA and multiplexed electrochemiluminescent assays. Multiparameter flow cytometry assessed the frequency, phenotype and proliferation of peripheral blood mononuclear cells. Preliminary antitumor activity was assessed by overall response rate (Response Evaluation Criteria in Solid Tumors V.1.1). RESULTS: As of March 2, 2020, median treatment duration was 7.5 weeks (range 1.1-77.1). Thirteen patients had discontinued and one (uveal melanoma) remains on treatment with stable disease. Best clinical response was stable disease (3 of 14 patients; 21%). The most frequent adverse events (AEs) were circular erythematous injection site reactions (100%), chills (71%), fatigue (57%), and fever (50%). Treatment-related grade 3/4 AEs occurred in six participants (43%); treatment-related serious AEs (SAEs) in three (21%). The per-protocol maximum tolerated dose was not reached. Pharmacokinetic accumulation of serum IL-15 in the first week was followed by significantly lower levels in week 2, likely due to more rapid cytokine consumption by an expanding lymphocyte pool. NIZ985 treatment was associated with increases in several cytokines, including interferon (IFN)-γ, IL-18, C-X-C motif chemokine ligand 10, and tumor necrosis factor-ß, plus significant induction of cytotoxic lymphocyte proliferation (including natural killer and CD8+ T cells), increased CD16+ monocytes, and increased CD163+ macrophages at injection sites. CONCLUSIONS: Subcutaneous NIZ985 TIW was generally well tolerated in patients with advanced cancer and produced immune activation paralleling preclinical observations, with induction of IFN-γ and proliferation of cytotoxic lymphocytes. Due to delayed SAEs at the two highest dose levels, administration is being changed to once-weekly in a revised protocol, as monotherapy and combined with checkpoint inhibitor spartalizumab. These alterations are expected to maximize the potential of NIZ985 as a novel immunotherapy. TRIAL REGISTRATION NUMBER: NCT02452268.

IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process.

IL-15 plays a pivotal role in the long-term survival of T cells and immunological memory. Its receptor consists of three subunits (IL-15Rα, IL-2/15Rß, and γc). IL-15 functions mainly via trans-presentation (TP), during which an APC expressing IL-15 bound to IL-15Rα presents the ligand to the ßγc receptor-heterodimer on a neighboring T/NK cell. To date, no direct biophysical evidence for the intercellular assembly of the IL-15R heterotrimer exists. Ag presentation (AP), the initial step of T cell activation, is also based on APC-T cell interaction. We were compelled to ask whether AP has any effect on IL-15 TP or whether they are independent processes. In our human Raji B cell-Jurkat T cell model system, we monitored inter-/intracellular protein interactions upon formation of IL-15 TP and AP receptor complexes by Förster resonance energy transfer measurements. We detected enrichment of IL-15Rα and IL-2/15Rß at the synapse and positive Förster resonance energy transfer efficiency if Raji cells were pretreated with IL-15, giving direct biophysical evidence for IL-15 TP. IL-15Rα and MHC class II interacted and translocated jointly to the immunological synapse when either ligand was present, whereas IL-2/15Rß and CD3 moved independently of each other. IL-15 TP initiated STAT5 phosphorylation in Jurkat cells, which was not further enhanced by AP. Conversely, IL-15 treatment slightly attenuated Ag-induced phosphorylation of the CD3ζ chain. Our studies prove that in our model system, IL-15 TP and AP can occur independently, and although AP enhances IL-15R assembly, it has no significant effect on IL-15 signaling during TP. Thus, IL-15 TP can be considered an autonomous, Ag-independent process.

Effective Cytotoxicity of Dendritic Cells against Established T Cell Lymphomas in Mice.

T cell lymphomas arise in mice that constitutively express a single TCR in the absence of NK cells. Upon TCR engagement these lymphomas are able to corrupt tumor surveillance by decreasing NK cell numbers. In this study, we investigate the outcome of interactions between these T cell lymphomas and dendritic cells. Bone marrow-derived dendritic cells mediated effective killing of T cell lymphomas after activation with IFN-γ and TLR ligands in culture. This cytotoxicity was independent of MHC compatibility. Cell lysis was reduced by the presence of the peroxynitrite inhibitors FeTTPS and L-NMMA, whereas inhibitors of apoptosis, death receptors, and degranulation were without effect, suggesting NO metabolites as the main mediators. When injected together with GM-CSF and R848 into lymphoma-bearing mice, in vitro-expanded bone marrow-derived dendritic cells caused significant survival increases. These data show that dendritic cell adaptive immunotherapy can be used as treatment against T cell lymphomas in mice.

Germinal epimutation of Fragile Histidine Triad (FHIT) gene is associated with progression to acute and chronic adult T-cell leukemia diseases.

BACKGROUND: Human T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation. METHODS: Here, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples. RESULTS: We found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients' samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation. CONCLUSIONS: These results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current "wait and see approach" in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

Immunometabolism at the Nexus of Cancer Therapeutic Efficacy and Resistance.

Constitutive activity of the immune surveillance system detects and kills cancerous cells, although many cancers have developed strategies to avoid detection and to resist their destruction. Cancer immunotherapy entails the manipulation of components of the endogenous immune system as targeted approaches to control and destroy cancer cells. Since one of the major limitations for the antitumor activity of immune cells is the immunosuppressive tumor microenvironment (TME), boosting the immune system to overcome the inhibition provided by the TME is a critical component of oncotherapeutics. In this article, we discuss the main effects of the TME on the metabolism and function of immune cells, and review emerging strategies to potentiate immune cell metabolism to promote antitumor effects either as monotherapeutics or in combination with conventional chemotherapy to optimize cancer management.

EGR1 Addiction in Diffuse Large B-cell Lymphoma.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell-like subtype (ABC) than germinal center B cell-like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. IMPLICATIONS: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Short-course IL-15 given as a continuous infusion led to a massive expansion of effective NK cells: implications for combination therapy with antitumor antibodies.

BACKGROUND: Full application of cytokines as oncoimmunotherapeutics requires identification of optimal regimens. Our initial effort with intravenous bolus recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions. Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10) provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8+ lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we shortened time of infusion. Treatment with rhIL-15 at a dose of 3-5 µg/kg as a 5-day continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while effector cell stimulation was comparable to the CIV-10 regimen. METHODS: Eleven patients with metastatic cancers were treated with rhIL-15 CIV-5, 3 µg (n=4), 4 µg (n=3), and 5 µg/kg/day (n=4) in a phase I dose-escalation study (April 6, 2012). RESULTS: Impressive expansions of NK cells were seen at all dose levels (mean 34-fold), including CD56bright NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8+ T cells (mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further testing. Cytolytic capacity of CD56bright and CD56dim NK cells was increased by interleukin-15 assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural killer group 2D-mediated cytotoxicity. The best response was stable disease. CONCLUSIONS: IL-15 administered as CIV-5 substantially expanded NK cells with increased cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their mechanism of action including alemtuzumab, obinutuzumab, avelumab, and mogamulizumab could benefit from those NK cell expansions and provide a promising therapeutic strategy. TRIAL REGISTRATION NUMBERS: NCT01572493, NCT03759184, NCT03905135, NCT04185220 and NCT02689453.

NF-κB-induced R-loop accumulation and DNA damage select for nucleotide excision repair deficiencies in adult T cell leukemia.

Constitutive NF-κB activation (NF-κBCA) confers survival and proliferation advantages to cancer cells and frequently occurs in T/B cell malignancies including adult T cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1). Counterintuitively, NF-κBCA by the HTLV-1 transactivator/oncoprotein Tax induces a senescence response, and HTLV-1 infections in culture mostly result in senescence or cell-cycle arrest due to NF-κBCA How NF-κBCA induces senescence, and how ATL cells maintain NF-κBCA and avert senescence, remain unclear. Here we report that NF-κBCA by Tax increases R-loop accumulation and DNA double-strand breaks, leading to senescence. R-loop reduction via RNase H1 overexpression, and short hairpin RNA silencing of two transcription-coupled nucleotide excision repair (TC-NER) endonucleases that are critical for R-loop excision-Xeroderma pigmentosum F (XPF) and XPG-attenuate Tax senescence, enabling HTLV-1-infected cells to proliferate. Our data indicate that ATL cells are often deficient in XPF, XPG, or both and are hypersensitive to ultraviolet irradiation. This TC-NER deficiency is found in all ATL types. Finally, ATL cells accumulate R-loops in abundance. Thus, TC-NER deficits are positively selected during HTLV-1 infection because they facilitate the outgrowth of infected cells initially and aid the proliferation of ATL cells with NF-κBCA later. We suggest that TC-NER deficits and excess R-loop accumulation represent specific vulnerabilities that may be targeted for ATL treatment.

Enhanced efficacy of JAK1 inhibitor with mTORC1/C2 targeting in smoldering/chronic adult T cell leukemia.

Adult T-cell leukemia (ATL) is an aggressive T-cell lymphoproliferative malignancy of regulatory T lymphocytes (Tregs), caused by human T-cell lymphotropic virus 1 (HTLV-1). Interleukin 2 receptor alpha (IL-2Rα) is expressed in the leukemic cells of smoldering/chronic ATL patients, leading to constitutive activation of the JAK/STAT pathway and spontaneous proliferation. The PI3K/AKT/mTOR pathway also plays a critical role in ATL cell survival and proliferation. We previously performed a high-throughput screen that demonstrated additive/synergistic activity of Ruxolitinib, a JAK1/2 inhibitor, with AZD8055, an mTORC1/C2 inhibitor. However, effects of unintended JAK2 inhibition with Ruxolitinib limits it therapeutic potential for ATL patients, which lead us to evaluate a JAK1-specific inhibitor. Here, we demonstrated that Upadacitinib, a JAK-1 inhibitor, inhibited the proliferation of cytokine-dependent ATL cell lines and the expression of p-STAT5. Combinations of Upadacitinib with either AZD8055 or Sapanisertib, mTORC1/C2 inhibitors, showed anti-proliferative effects against cytokine-dependent ATL cell lines and synergistic effect with reducing tumor growth in NSG mice bearing IL-2 transgenic tumors. Importantly, the combination of these two agents inhibited ex vivo spontaneous proliferation of ATL cells from patients with smoldering/chronic ATL. Combined targeting of JAK/STAT and PI3K/AKT/mTOR pathways represents a promising therapeutic intervention for patients with smoldering/chronic ATL.