Therapeutic application of human type 2 innate lymphoid cells via induction of granzyme B-mediated tumor cell death.

The therapeutic potential for human type 2 innate lymphoid cells (ILC2s) has been underexplored. Although not observed in mouse ILC2s, we found that human ILC2s secrete granzyme B (GZMB) and directly lyse tumor cells by inducing pyroptosis and/or apoptosis, which is governed by a DNAM-1-CD112/CD155 interaction that inactivates the negative regulator FOXO1. Over time, the high surface density expression of CD155 in acute myeloid leukemia cells impairs the expression of DNAM-1 and GZMB, thus allowing for immune evasion. We describe a reliable platform capable of up to 2,000-fold expansion of human ILC2s within 4 weeks, whose molecular and cellular ILC2 profiles were validated by single-cell RNA sequencing. In both leukemia and solid tumor models, exogenously administered expanded human ILC2s show significant antitumor effects in vivo. Collectively, we demonstrate previously unreported properties of human ILC2s and identify this innate immune cell subset as a member of the cytolytic immune effector cell family.

ILC1s control leukemia stem cell fate and limit development of AML.

Type I innate lymphoid cells (ILC1s) are critical regulators of inflammation and immunity in mammalian tissues. However, their function in cancer is mostly undefined. Here, we show that a high density of ILC1s induces leukemia stem cell (LSC) apoptosis in mice. At a lower density, ILC1s prevent LSCs from differentiating into leukemia progenitors and promote their differentiation into non-leukemic cells, thus blocking the production of terminal myeloid blasts. All of these effects, which require ILC1s to produce interferon-γ after cell-cell contact with LSCs, converge to suppress leukemogenesis in vivo. Conversely, the antileukemia potential of ILC1s wanes when JAK-STAT or PI3K-AKT signaling is inhibited. The relevant antileukemic properties of ILC1s are also functional in healthy individuals and impaired in individuals with acute myeloid leukemia (AML). Collectively, these findings identify ILC1s as anticancer immune cells that might be suitable for AML immunotherapy and provide a potential strategy to treat AML and prevent relapse of the disease.

R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis.

R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.

Homoharringtonine Inhibits the NOTCH/MYC Pathway and Exhibits Anti-Tumor Effects in T-Cell Acute Lymphoblastic Leukemia.

We report on the antileukemic activity of homoharringtonine (HHT) in T-ALL. We showed that HHT inhibited NOTCH/MYC pathway and induced a significantly longer survival in T-ALL mouse and patient-derived xenograft models, therefore supporting HHT as a promising agent for T-ALL.

A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ-induced CD38 expression.

ABSTRACT: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

Glofitamab stimulates immune cell infiltration of CNS tumors and induces clinical responses in secondary CNS lymphoma.

ABSTRACT: Although CD20×CD3 bispecific antibodies are effective against systemic B-cell lymphomas, their efficacy in central nervous system (CNS) lymphoma is unknown. Here, we report the CD20×CD3 bispecific glofitamab penetrates the blood-brain barrier, stimulates immune-cell infiltration of CNS tumors, and induces clinical responses in patients with secondary CNS.

miR-328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts.

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

MicroRNA networks in FLT3-ITD acute myeloid leukemia.

MiR-126 and miR-155 are key microRNAs (miRNAs) that regulate, respectively, hematopoietic cell quiescence and proliferation. Herein we showed that in acute myeloid leukemia (AML), the biogenesis of these two miRNAs is interconnected through a network of regulatory loops driven by the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). In fact, FLT3-ITD induces the expression of miR-155 through a noncanonical mechanism of miRNA biogenesis that implicates cytoplasmic Drosha ribonuclease III (DROSHA). In turn, miR-155 down-regulates SH2-containing inositol phosphatase 1 (SHIP1), thereby increasing phosphor-protein kinase B (AKT) that in turn serine-phosphorylates, stabilizes, and activates Sprouty related EVH1 domain containing 1 (SPRED1). Activated SPRED1 inhibits the RAN/XPO5 complex and blocks the nucleus-to-cytoplasm transport of pre-miR-126, which cannot then complete the last steps of biogenesis. The net result is aberrantly low levels of mature miR-126 that allow quiescent leukemia blasts to be recruited into the cell cycle and proliferate. Thus, miR-126 down-regulation in proliferating AML blasts is downstream of FLT3-ITD­dependent miR-155 expression that initiates a complex circuit of concatenated regulatory feedback (i.e., miR-126/SPRED1, miR-155/human dead-box protein 3 [DDX3X]) and feed-forward (i.e., miR-155/SHIP1/AKT/miR-126) regulatory loops that eventually converge into an output signal for leukemic growth.

A phase 1 trial of 8-chloro-adenosine in relapsed/refractory acute myeloid leukemia: An evaluation of safety and pharmacokinetics.

BACKGROUND: This study evaluated the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of 8-chloro-adenosine (8-Cl-Ado) in patients with relapsed/refractory acute myeloid leukemia (AML). METHODS: 8-Cl-Ado was administered daily for 5 days; the starting dose was 100 mg/m2 , the highest dose tested was 800 mg/m2 . The end points were toxicity, disease response, and PK/PD measurements. RESULTS: The predominant nonhematologic toxicity was cardiac with grade ≥3 toxicity. Plasma PK in all patients suggested heterogeneity among patients, yet, some dose-dependency for the accumulation of 8-Cl-Ado. Two 8-Cl-Ado metabolites accumulated at similar levels to 8-Cl-Ado. Cellular PK in eight patients indicated accumulation of 8-Cl-ATP, which was associated with AML blast cytoreduction in peripheral blood. The authors determined the RP2D of 8-Cl-Ado to be 400 mg/m2 . CONCLUSIONS: Given the cardiac adverse events observed, patients require monitoring for arrhythmias and QT interval during infusion. Although peripheral blood cytoreduction was observed, responses were transient, suggesting combination strategies will be required.

Therapy-related acute lymphoblastic leukaemia in women with antecedent breast cancer.

Therapy-related acute lymphoblastic leukaemia (tr-ALL) is a disease entity attributed to previous exposure to chemotherapy and/or radiation for antecedent malignancy. There is observed female predominance for tr-ALL, likely due to high prevalence and excellent curable rate for non-metastatic breast cancer as well as the frequent use of carcinogenic agents as part of adjuvant therapy. Here, we reviewed 37 women with diagnosis of ALL following breast cancer treatment with focus on cytogenetic categorization. Philadelphia chromosome positivity (Ph+), KMT2A alterations and other cytogenetic change groups were observed in 32%, 22% and 46% of patients respectively. Median overall survival (OS) and relapse-free survival (RFS) were 19.4 and 12.9 months, overall while both OS and RFS were superior in tr-ALL with Ph+ disease compared to KMT2Ar and other cytogenetics respectively. Seventeen (45.9%) patients underwent consolidative allogeneic haematopoietic cell transplantation (alloHCT) in CR1 out of which 4 (24%) relapsed following transplant. Both OS and RFS were superior in the KMT2Ar cytogenetics group following alloHCT. Ph chromosome represents the largest genetic entity of tr-ALL following breast cancer therapy, and it may be associated with superior survival outcomes while KMT2Ar may be associated with poorer outcomes that can perhaps be mitigated by alloHSCT.

Disruption of dNTP homeostasis by ribonucleotide reductase hyperactivation overcomes AML differentiation blockade.

Differentiation blockade is a hallmark of acute myeloid leukemia (AML). A strategy to overcome such a blockade is a promising approach against the disease. The lack of understanding of the underlying mechanisms hampers development of such strategies. Dysregulated ribonucleotide reductase (RNR) is considered a druggable target in proliferative cancers susceptible to deoxynucleoside triphosphate (dNTP) depletion. Herein, we report an unanticipated discovery that hyperactivating RNR enables differentiation and decreases leukemia cell growth. We integrate pharmacogenomics and metabolomics analyses to identify that pharmacologically (eg, nelarabine) or genetically upregulating RNR subunit M2 (RRM2) creates a dNTP pool imbalance and overcomes differentiation arrest. Moreover, R-loop-mediated DNA replication stress signaling is responsible for RRM2 activation by nelarabine treatment. Further aggravating dNTP imbalance by depleting the dNTP hydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) enhances ablation of leukemia stem cells by RRM2 hyperactivation. Mechanistically, excessive activation of extracellular signal-regulated kinase (ERK) signaling downstream of the imbalance contributes to cellular outcomes of RNR hyperactivation. A CRISPR screen identifies a synthetic lethal interaction between loss of DUSP6, an ERK-negative regulator, and nelarabine treatment. These data demonstrate that dNTP homeostasis governs leukemia maintenance, and a combination of DUSP inhibition and nelarabine represents a therapeutic strategy.

Blinatumomab-induced macrophage activating syndrome (MAS) in adult with B-cell acute lymphoblastic leukemia (B-ALL).

Blinatumomab as a single agent has demonstrated superiority over salvage chemotherapy in patients with relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL), with manageable safety and efficacy. Though known to have anticipated drug toxicities including cytokine release syndrome (CRS) and neurotoxicity, there is only one prior report of macrophage activating syndrome (MAS) due to blinatumomab. Case Presentation: We report the first case of blinatumomab-induced MAS in an adult. The patient presented with fever, cough, and weakness on the second cycle of blinatumomab. Complete blood count was notable for severe leukopenia, with comprehensive metabolic panel notable for elevated alkaline phosphatase, AST, ALT, LDH, and hyperferritinemia consistent with MAS. The patient was already in MRD-negative remission at presentation with MAS. She responded rapidly to withholding the drug and administration of both tocilizumab and dexamethasone. She was able to restart therapy with blinatumomab dosed at 9 mcg/day with no recurrence of symptoms. Though MAS is not an expected association with blinatumomab, the risk for CRS is. Secondary MAS in this case likely shares a mechanism with other hyperinflammatory conditions. Management includes holding the offending agent, like blinatumomab, and administering tocilizumab and dexamethasone. Future research will be needed to predict which patients are at highest risk to develop MAS after similar T-cell therapies.

Outcomes following allogeneic hematopoietic cell transplantation relapse in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Improved first progression-free survival following allogeneic hematopoietic cell transplantation relapse with the use of immunotherapy.

Toxicities associated with tyrosine kinase inhibitor maintenance following allogeneic hematopoietic cell transplantation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Allogeneic hematopoietic cell transplantation (HCT) offers a potential cure in Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL); nonetheless, relapses are common and the major cause of mortality. One strategy to prevent relapse is tyrosine kinase inhibitor (TKI) maintenance post-HCT, but published clinical experience is primarily with the first-generation TKI imatinib while data with newer generation TKIs are limited. We conducted a retrospective analysis of 185 Ph+ ALL patients who underwent HCT followed by TKI maintenance from 2003 to 2021 at City of Hope. Initially, 50 (27.0%) received imatinib, 118 (63.8%) received a second-generation TKI (2G-TKI), and 17 (9.2%) received ponatinib. A total of 77 patients (41.6%) required a dose reduction of their initial TKI due to toxicity. Sixty-six patients (35.7%) did not complete maintenance due to toxicity; 69 patients (37.3%) discontinued 1 TKI, and 11 (5.9%) discontinued 2 TKIs due to toxicity. Initial imatinib versus 2G-TKI versus ponatinib maintenance was discontinued in 19 (38.0%) versus 68 (57.6%) versus 3 (17.6%) patients due to toxicity (p = .003), respectively. Patients on ponatinib as their initial TKI had a longer duration of TKI maintenance versus 2G-TKI: 576.0 days (range, 72-921) versus 254.5 days (range, 3-2740; p = .02). The most common reasons for initial TKI discontinuation include gastrointestinal (GI) intolerance (15.1%), cytopenia (8.6%), and fluid retention (3.8%). The 5-year overall survival and progression-free survival for the total population were 78% and 71%, respectively. Our findings demonstrate the challenges of delivering post-HCT TKI maintenance in a large real-world cohort as toxicities leading to TKI interruptions, discontinuation, and dose reduction were common.

Concurrent versus sequential or no triazole anti-fungal therapy in patients undergoing 7 + 3 plus midostaurin induction for FLT-3 acute myelogenous leukemia.

INTRODUCTION: Midostaurin is a multikinase inhibitor approved for the treatment of adult patients with newly diagnosed FMS-like tyrosine kinase 3 mutated (FLT3m) acute myeloid leukemia (AML). Azole antifungal medications are commonly used in AML and are known to interact with anti-cancer drugs such as midostaurin through the CYP3A pathway. However, there are no midostaurin related dose modifications recommended with strong CYP3A inhibitors. METHODS: We retrospectively reviewed 40 patients between 2017-2022 and compared efficacy and safety outcomes in patients who received azole antifungals concurrently to those who did not receive an azole or received it sequentially to midostaurin for treatment of FLT3m AML. RESULTS: Median age of both groups was approximately 55 years and 70% of patients harbored FLT-3 internal tandem duplication mutations. Most patients in the concurrent arm were on either posaconazole (33%) or isavuconazole (50%) for antifungal prophylaxis and micafungin (72%) for the sequential/no azole arm. Overall CR/CRi rate with concurrent versus sequential/no azole were 72% and 77%, and non-hematologic grade 3 toxicities were 22% and 40% (p = 0.21), respectively. Rates of dose reductions (6% vs. 0%, p = 0.26) and held doses (17% vs. 14%, p = 0.79) were not different between concurrent and sequential/no azole. There were no differences in the rates of new fungal infection during induction between the two groups. CONCLUSION: Azoles given concurrently or sequentially with midostaurin were found to be equally safe and effective in the treatment of newly diagnosed FLT3 AML. Additional confirmatory studies are needed due to our limited sample size.

BRAF Mutations in Patients with Myeloid Neoplasms: A Cancer Center Multigene Next-Generation Sequencing Analysis Experience.

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.

Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) has a poor survival rate for both pediatric and adult patients due to its frequent relapse. To elucidate the bioenergetic principle underlying AML relapse, we investigated the transcriptional regulation of mitochondrial-nuclear dual genomes responsible for metabolic plasticity in treatment-resistant blasts. Both the gain and loss of function results demonstrated that NFκB2, a noncanonical transcription factor (TF) of the NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) family, can control the expression of TFAM (mitochondrial transcription factor A), which is known to be essential for metabolic biogenesis. Furthermore, genetic tracking and promoter assays revealed that NFκB2 is in the mitochondria and can bind the specific "TTGGGGGGTG" region of the regulatory D-loop domain to activate the light-strand promoter (LSP) and heavy-strand promoter 1 (HSP1), promoters of the mitochondrial genome. Based on our discovery of NFκB2's novel function of regulating mitochondrial-nuclear dual genomes, we explored a novel triplet therapy including inhibitors of NFκB2, tyrosine kinase, and mitochondrial ATP synthase that effectively eliminated primary AML blasts with mutations of the FMS-related receptor tyrosine kinase 3 (FLT3) and displayed minimum toxicity to control cells ex vivo. As such, effective treatments for AML must include strong inhibitory actions on the dual genomes mediating metabolic plasticity to improve leukemia prognosis.

Activated natural killer cells predict poor clinical prognosis in high-risk B- and T-cell acute lymphoblastic leukemia.

B- and T-cell acute lymphoblastic leukemia (B/T-ALL) may be refractory or recur after therapy by suppressing host anticancer immune surveillance mediated specifically by natural killer (NK) cells. We delineated the phenotypic and functional defects in NK cells from high-risk patients with B/T-ALL using mass cytometry, flow cytometry, and in silico cytometry, with the goal of further elucidating the role of NK cells in sustaining acute lymphoblastic leukemia (ALL) regression. We found that, compared with their normal counterparts, NK cells from patients with B/T-ALL are less cytotoxic but exhibit an activated signature that is characterized by high CD56, high CD69, production of activated NK cell-origin cytokines, and calcium (Ca2+) signaling. We demonstrated that defective maturation of NK cells into cytotoxic effectors prevents NK cells from ALL from lysing NK cell-sensitive targets as efficiently as do normal NK cells. Additionally, we showed that NK cells in ALL are exhausted, which is likely caused by their chronic activation. We found that increased frequencies of activated cytokine-producing NK cells are associated with increased disease severity and independently predict poor clinical outcome in patients with ALL. Our studies highlight the benefits of developing NK cell profiling as a diagnostic tool to predict clinical outcome in patients with ALL and underscore the clinical potential of allogeneic NK cell infusions to prevent ALL recurrence.