BATF is a major driver of NK cell epigenetic reprogramming and dysfunction in AML.

Myelodysplastic syndrome and acute myeloid leukemia (AML) belong to a continuous disease spectrum of myeloid malignancies with poor prognosis in the relapsed/refractory setting necessitating novel therapies. Natural killer (NK) cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and an exhausted phenotype at the single-cell transcriptomic and proteomic levels. In this study, we identified that this dysfunction was mediated through a cross-talk between NK cells and myeloid blasts necessitating cell-cell contact. NK cell dysfunction could be prevented by targeting the αvß-integrin/TGF-ß/SMAD pathway but, once established, was persistent because of profound epigenetic reprogramming. We identified BATF as a core transcription factor and the main mediator of this NK cell dysfunction in AML. Mechanistically, we found that BATF was directly regulated and induced by SMAD2/3 and, in turn, bound to key genes related to NK cell exhaustion, such as HAVCR2, LAG3, TIGIT, and CTLA4. BATF deletion enhanced NK cell function against AML in vitro and in vivo. Collectively, our findings reveal a previously unidentified mechanism of NK immune evasion in AML manifested by epigenetic rewiring and inactivation of NK cells by myeloid blasts. This work highlights the importance of using healthy allogeneic NK cells as an adoptive cell therapy to treat patients with myeloid malignancies combined with strategies aimed at preventing the dysfunction by targeting the TGF-ß pathway or BATF.

CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

Comparison of two autologous hematopoietic stem cell mobilization strategies in patients with multiple myeloma: CE plus G-CSF versus G-CSF only: A single-center retrospective analysis.

BACKGROUND: Despite recent advances in the treatment of multiple myeloma, high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) remains an essential therapeutic keystone. As for the stem cell mobilization procedure, different regimens have been established, usually consisting of a cycle of chemotherapy followed by application of granulocyte-colony stimulating factor (G-CSF), although febrile neutropenia is a common complication. Following national guidelines, our institution decided to primarily use G-CSF only mobilization during the COVID-19 pandemic to minimize the patients' risk of infection and to reduce the burden on the health system. STUDY DESIGN AND METHODS: In this retrospective single-center analysis, the efficacy and safety of G-CSF only mobilization was evaluated and compared to a historic control cohort undergoing chemotherapy-based mobilization by cyclophosphamide and etoposide (CE) plus G-CSF. RESULTS: Although G-CSF only was associated with a higher need for plerixafor administration (p < .0001) and a higher number of apheresis sessions per patient (p = .0002), we were able to collect the target dose of hematopoietic stem cells in the majority of our patients. CE mobilization achieved higher hematopoietic stem cell yields (p = .0015) and shorter apheresis sessions (p < .0001) yet was accompanied by an increased risk of febrile neutropenia (p < .0001). There was no difference in engraftment after ASCT. DISCUSSION: G-CSF only mobilization is a useful option in selected patients with comorbidities and an increased risk of serious infections, especially in the wintertime or in future pandemics.

How I treat high-risk acute myeloid leukemia using preemptive adoptive cellular immunotherapy.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for patients with high-risk acute leukemias, but unfortunately disease recurrence remains the major cause of death in these patients. Infusion of donor lymphocytes (DLI) has the potential to restore graft-versus-leukemia immunologic surveillance; however, efficacy varies across different hematologic entities. Although relapsed chronic myeloid leukemia, transplanted in chronic phase, has proven remarkably susceptible to DLI, response rates are more modest for relapsed acute myeloid leukemia and acute lymphoblastic leukemia. To prevent impending relapse, a number of groups have explored administering DLI preemptively on detection of measurable residual disease (MRD) or mixed chimerism. Evidence for the effectiveness of this strategy, although encouraging, comes from only a few, mostly single-center retrospective, nonrandomized studies. This article seeks to (1) discuss the available evidence supporting this approach while highlighting some of the inherent challenges of MRD-triggered treatment decisions post-transplant, (2) portray other forms of postremission cellular therapies, including the role of next-generation target-specific immunotherapies, and (3) provide a practical framework to support clinicians in their decision-making process when considering preemptive cellular therapy for this difficult-to-treat patient population.

Multiplexed engineering and precision gene editing in cellular immunotherapy.

The advent of cellular immunotherapy in the clinic has entirely redrawn the treatment landscape for a growing number of human cancers. Genetically reprogrammed immune cells, including chimeric antigen receptor (CAR)-modified immune effector cells as well as T cell receptor (TCR) therapy, have demonstrated remarkable responses across different hard-to-treat patient populations. While these novel treatment options have had tremendous success in providing long-term remissions for a considerable fraction of treated patients, a number of challenges remain. Limited in vivo persistence and functional exhaustion of infused immune cells as well as tumor immune escape and on-target off-tumor toxicities are just some examples of the challenges which restrain the potency of today's genetically engineered cell products. Multiple engineering strategies are being explored to tackle these challenges.The advent of multiplexed precision genome editing has in recent years provided a flexible and highly modular toolkit to specifically address some of these challenges by targeted genetic interventions. This class of next-generation cellular therapeutics aims to endow engineered immune cells with enhanced functionality and shield them from immunosuppressive cues arising from intrinsic immune checkpoints as well as the hostile tumor microenvironment (TME). Previous efforts to introduce additional genetic modifications into immune cells have in large parts focused on nuclease-based tools like the CRISPR/Cas9 system or TALEN. However, nuclease-inactive platforms including base and prime editors have recently emerged and promise a potentially safer route to rewriting genetic sequences and introducing large segments of transgenic DNA without inducing double-strand breaks (DSBs). In this review, we discuss how these two exciting and emerging fields-cellular immunotherapy and precision genome editing-have co-evolved to enable a dramatic expansion in the possibilities to engineer personalized anti-cancer treatments. We will lay out how various engineering strategies in addition to nuclease-dependent and nuclease-inactive precision genome editing toolkits are increasingly being applied to overcome today's limitations to build more potent cellular therapeutics. We will reflect on how novel information-rich unbiased discovery approaches are continuously deepening our understanding of fundamental mechanisms governing tumor biology. We will conclude with a perspective of how multiplexed-engineered and gene edited cell products may upend today's treatment paradigms as they evolve into the next generation of more potent cellular immunotherapies.

KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape.

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

Natural killer cells in antitumour adoptive cell immunotherapy.

Natural killer (NK) cells comprise a unique population of innate lymphoid cells endowed with intrinsic abilities to identify and eliminate virally infected cells and tumour cells. Possessing multiple cytotoxicity mechanisms and the ability to modulate the immune response through cytokine production, NK cells play a pivotal role in anticancer immunity. This role was elucidated nearly two decades ago, when NK cells, used as immunotherapeutic agents, showed safety and efficacy in the treatment of patients with advanced-stage leukaemia. In recent years, following the paradigm-shifting successes of chimeric antigen receptor (CAR)-engineered adoptive T cell therapy and the advancement in technologies that can turn cells into powerful antitumour weapons, the interest in NK cells as a candidate for immunotherapy has grown exponentially. Strategies for the development of NK cell-based therapies focus on enhancing NK cell potency and persistence through co-stimulatory signalling, checkpoint inhibition and cytokine armouring, and aim to redirect NK cell specificity to the tumour through expression of CAR or the use of engager molecules. In the clinic, the first generation of NK cell therapies have delivered promising results, showing encouraging efficacy and remarkable safety, thus driving great enthusiasm for continued innovation. In this Review, we describe the various approaches to augment NK cell cytotoxicity and longevity, evaluate challenges and opportunities, and reflect on how lessons learned from the clinic will guide the design of next-generation NK cell products that will address the unique complexities of each cancer.

Engineering the next generation of CAR-NK immunotherapies.

Over the past few years, cellular immunotherapy has emerged as a novel treatment option for certain forms of hematologic malignancies with multiple CAR-T therapies now routinely administered in the clinic. The limitations of generating an autologous cell product and the challenges of toxicity with CAR-T cells underscore the need to develop novel cell therapy products that are universal, safe, and potent. Natural killer (NK) cells are part of the innate immune system with unique advantages, including the potential for off-the-shelf therapy. A recent first-in-human trial of CD19-CAR-NK infusion in patients with relapsed/refractory lymphoid malignancies proved safe with promising clinical activity. Building on these encouraging clinical responses, research is now actively exploring ways to further enhance CAR-NK cell potency by prolonging in vivo persistence and overcoming mechanisms of functional exhaustion. Besides these strategies to modulate CAR-NK cell intrinsic properties, there are increasing efforts to translate the successes seen in hematologic malignancies to the solid tumor space. This review will provide an overview on current trends and evolving concepts to genetically engineer the next generation of CAR-NK therapies. Emphasis will be placed on innovative multiplexed engineering approaches including CRISPR/Cas9 to overcome CAR-NK functional exhaustion and reprogram immune cell metabolism for enhanced potency.

RACE-trial: neoadjuvant radiochemotherapy versus chemotherapy for patients with locally advanced, potentially resectable adenocarcinoma of the gastroesophageal junction - a randomized phase III joint study of the AIO, ARO and DGAV.

BACKGROUND: Despite obvious advances over the last decades, locally advanced adenocarcinomas of the gastroesophageal junction (GEJ) still carry a dismal prognosis with overall 5-year survival rates of less than 50% even when using modern optimized treatment protocols such as perioperative chemotherapy based on the FLOT regimen or radiochemotherapy. Therefore the question remains whether neoadjuvant chemotherapy or neoadjuvant radiochemotherapy is eliciting the best results in patients with GEJ cancer. Hence, an adequately powered multicentre trial comparing both therapeutic strategies is clearly warranted. METHODS: The RACE trial is a an investigator initiated multicenter, prospective, randomized, stratified phase III clinical trial and seeks to investigate the role of preoperative induction chemotherapy (2 cycles of FLOT: 5-FU, leucovorin, oxaliplatin, docetaxel) with subsequent preoperative radiochemotherapy (oxaliplatin weekly, 5-FU plus concurrent fractioned radiotherapy to a dose of 45 Gy) compared to preoperative chemotherapy alone (4 cycles of FLOT), both followed by resection and postoperative completion of chemotherapy (4 cycles of FLOT), in the treatment of locally advanced, potentially resectable adenocarcinoma of the gastroesophageal junction. Patients with cT3-4, any N, M0 or cT2 N+, M0 adenocarcinoma of the GEJ are eligible for inclusion. The RACE trial aims to enrol 340 patients to be allocated to both treatment arms in a 1:1 ratio stratified by tumour site. The primary endpoint of the trial is progression-free survival assessed with follow-up of maximum 60 months. Secondary endpoints include overall survival, R0 resection rate, number of harvested lymph nodes, site of tumour relapse, perioperative morbidity and mortality, safety and toxicity and quality of life. DISCUSSION: The RACE trial compares induction chemotherapy with FLOT followed by preoperative oxaliplatin and 5-Fluorouracil-based chemoradiation versus preoperative chemotherapy with FLOT alone, both followed by surgery and postoperative completion of FLOT chemotherapy in the treatment of locally advanced, non-metastatic adenocarcinoma of the GEJ. The trial aims to show superiority of the combined chemotherapy/radiochemotherapy treatment, assessed by progression-free survival, over perioperative chemotherapy alone. TRIAL REGISTRATION: ClinicalTrials.gov ; NCT04375605 ; Registered 4th May 2020.

SUMO pathway inhibition targets an aggressive pancreatic cancer subtype.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) still carries a dismal prognosis with an overall 5-year survival rate of 9%. Conventional combination chemotherapies are a clear advance in the treatment of PDAC; however, subtypes of the disease exist, which exhibit extensive resistance to such therapies. Genomic MYC amplifications represent a distinct subset of PDAC with an aggressive tumour biology. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. The aim of the study was to find and to target MYC-associated dependencies. DESIGN: We analysed human PDAC gene expression datasets. Results were corroborated by the analysis of the small ubiquitin-like modifier (SUMO) pathway in a large PDAC cohort using immunohistochemistry. A SUMO inhibitor was used and characterised using human and murine two-dimensional, organoid and in vivo models of PDAC. RESULTS: We observed that MYC is connected to the SUMOylation machinery in PDAC. Components of the SUMO pathway characterise a PDAC subtype with a dismal prognosis and we provide evidence that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition. CONCLUSION: SUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype.