IL-9 secreted by leukemia stem cells induces Th1-skewed CD4+ T cells, which promote their expansion.

ABSTRACT: In acute myeloid leukemia (AML), leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) interact with various cell types in the bone marrow (BM) microenvironment, regulating their expansion and differentiation. To study the interaction of CD4+ and CD8+ T cells in the BM with LSCs and LPCs, we analyzed their transcriptome and predicted cell-cell interactions by unbiased high-throughput correlation network analysis. We found that CD4+ T cells in the BM of patients with AML were activated and skewed toward T-helper (Th)1 polarization, whereas interleukin-9 (IL-9)-producing (Th9) CD4+ T cells were absent. In contrast to normal hematopoietic stem cells, LSCs produced IL-9, and the correlation modeling predicted IL9 in LSCs as a main hub gene that activates CD4+ T cells in AML. Functional validation revealed that IL-9 receptor signaling in CD4+ T cells leads to activation of the JAK-STAT pathway that induces the upregulation of KMT2A and KMT2C genes, resulting in methylation on histone H3 at lysine 4 to promote genome accessibility and transcriptional activation. This induced Th1-skewing, proliferation, and effector cytokine secretion, including interferon gamma (IFN-γ) and tumor necrosis factor α (TNF-α). IFN-γ and, to a lesser extent, TNF-α produced by activated CD4+ T cells induced the expansion of LSCs. In accordance with our findings, high IL9 expression in LSCs and high IL9R, TNF, and IFNG expression in BM-infiltrating CD4+ T cells correlated with worse overall survival in AML. Thus, IL-9 secreted by AML LSCs shapes a Th1-skewed immune environment that promotes their expansion by secreting IFN-γ and TNF-α.

A phase Ib/II randomized, open-label drug repurposing trial of glutamate signaling inhibitors in combination with chemoradiotherapy in patients with newly diagnosed glioblastoma: the GLUGLIO trial protocol.

BACKGROUND: Glioblastoma is the most common and most aggressive malignant primary brain tumor in adults. Glioblastoma cells synthesize and secrete large quantities of the excitatory neurotransmitter glutamate, driving epilepsy, neuronal death, tumor growth and invasion. Moreover, neuronal networks interconnect with glioblastoma cell networks through glutamatergic neuroglial synapses, activation of which induces oncogenic calcium oscillations that are propagated via gap junctions between tumor cells. The primary objective of this study is to explore the efficacy of brain-penetrating anti-glutamatergic drugs to standard chemoradiotherapy in patients with glioblastoma. METHODS/DESIGN: GLUGLIO is a 1:1 randomized phase Ib/II, parallel-group, open-label, multicenter trial of gabapentin, sulfasalazine, memantine and chemoradiotherapy (Arm A) versus chemoradiotherapy alone (Arm B) in patients with newly diagnosed glioblastoma. Planned accrual is 120 patients. The primary endpoint is progression-free survival at 6 months. Secondary endpoints include overall and seizure-free survival, quality of life of patients and caregivers, symptom burden and cognitive functioning. Glutamate levels will be assessed longitudinally by magnetic resonance spectroscopy. Other outcomes of interest include imaging response rate, neuronal hyperexcitability determined by longitudinal electroencephalography, Karnofsky performance status as a global measure of overall performance, anticonvulsant drug use and steroid use. Tumor tissue and blood will be collected for translational research. Subgroup survival analyses by baseline parameters include segregation by age, extent of resection, Karnofsky performance status, O6-methylguanine DNA methyltransferase (MGMT) promotor methylation status, steroid intake, presence or absence of seizures, tumor volume and glutamate levels determined by MR spectroscopy. The trial is currently recruiting in seven centers in Switzerland. TRIAL REGISTRATION: NCT05664464. Registered 23 December 2022.

Results from a phase I/II trial of cusatuzumab combined with azacitidine in patients with newly diagnosed acute myeloid leukemia who are ineligible for intensive chemotherapy.

Cusatuzumab is a high-affinity, anti-CD70 monoclonal antibody under investigation in acute myeloid leukemia (AML). This two-part, open-label, multicenter, phase I/II trial evaluated cusatuzumab plus azacitidine in patients with newly diagnosed AML ineligible for intensive chemotherapy. Patients received a single dose of cusatuzumab at one of four dose levels (1, 3, 10, or 20 mg/kg) 14 days before starting combination therapy. In phase I dose escalation, cusatuzumab was then administered on days 3 and 17, in combination with azacitidine (75 mg/m2) on days 1-7, every 28 days. The primary objective in phase I was to determine the recommended phase II dose (RP2D) of cusatuzumab plus azacitidine. The primary objective in phase II was efficacy at the RP2D (selected as 10 mg/kg). Thirty-eight patients were enrolled: 12 in phase I (three per dose level; four with European LeukemiaNet 2017 adverse risk) and 26 in phase II (21 with adverse risk). An objective response (≥partial remission) was achieved by 19/38 patients (including 8/26 in phase II); 14/38 achieved complete remission. Eleven patients (37.9%) achieved an objective response among the 29 patients in phase I and phase II treated at the RP2D. At a median follow-up of 10.9 months, median duration of first response was 4.5 months and median overall survival was 11.5 months. The most common treatment-emergent adverse events were infections (84.2%) and hematologic toxicities (78.9%). Seven patients (18.4%) reported infusion-related reactions, including two with grade 3 events. Thus, cusatuzumab/azacitidine appears generally well tolerated and shows preliminary efficacy in this setting. Investigation of cusatuzumab combined with current standard-of-care therapy, comprising venetoclax and azacitidine, is ongoing.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

The effect of neoadjuvant therapy on PD-L1 expression and CD8+lymphocyte density in non-small cell lung cancer.

PD-L1 expression is the routine clinical biomarker for the selection of patients to receive immunotherapy in non-small cell lung cancer (NSCLC). However, the application and best timing of immunotherapy in the resectable setting is still under investigation. We aimed to study the effect of chemotherapy on PD-L1 expression and tumor infiltrating lymphocytes (TILs), which is to date still poorly understood. Our retrospective, single-centre neoadjuvant cohort comprised 96 consecutive patients with NSCLC resected 2000-2016 after neoadjuvant therapy, including paired diagnostic chemo-naïve specimens in 53 cases. A biologically matched surgical cohort of 114 primary resected cases was included. PD-L1 expression, CD8 + TILs density and tertiary lymphoid structures were assessed on whole slides and correlated with clinico-pathological characteristics and survival. Seven/53 and 12/53 cases had lower respectively higher PD-L1 expressions after neoadjuvant therapy. Most cases (n = 34) showed no changes in PD-L1 expression, the majority of these harboring PD-L1 < 1% in both samples (21/34 [61.8%]). Although CD8 + TILs density was significantly higher after chemotherapy (p = 0.031) in resections compared to diagnostic biopsies, this might be due to sampling and statistical bias. No difference in PD-L1 expression or CD8 + TILs density was detected when comparing the neoadjuvant and surgical cohort. In univariable analyses, higher CD8 + TILs density, higher numbers of tertiary lymphoid structures but not PD-L1 expression were significantly associated with longer survival. Increased PD-L1 expression after neoadjuvant chemotherapy was not significantly associated with shorter 5-year survival, but the number of cases was very low. In multivariable analysis, only pT category and age remained independent prognostic factors. In summary, PD-L1 expression was mostly unchanged after neoadjuvant chemotherapy compared to diagnostic biopsies. The sample size of cases with changed PD-L1 expression was too small to draw conclusions on any prognostic value.

A prognostic score for non-small cell lung cancer resected after neoadjuvant therapy in comparison with the tumor-node-metastases classification and major pathological response.

Studies validating the prognostic accuracy of the tumor-node-metastases (TNM) classification in patients with lung cancer treated by neoadjuvant therapy are scarce. Tumor regression, particularly major pathological response (MPR), is an acknowledged prognostic factor in this setting. We aimed to validate a novel combined prognostic score. This retrospective single-center study was conducted on 117 consecutive patients with non-small cell lung cancer resected after neoadjuvant treatment at a Swiss University Cancer Center between 2000 and 2016. All cases were clinicopathologically re-evaluated. We assessed the prognostic performance of a novel prognostic score (PRSC) combining T-category, lymph node status, and MPR, in comparison with the eighth edition of the TNM classification (TNM8), the size adapted TNM8 as proposed by the International Association for the Study of Lung Cancer (IASLC) and MPR alone. The isolated ypT-category and the combined TNM8 stages accurately differentiated overall survival (OS, stage p = 0.004) and disease-free survival (DFS, stage p = 0.018). Tumor regression had a prognostic impact. Optimal cut-offs for MPR emerged as 65% for adenocarcinoma and 10% for non-adenocarcinoma and were statistically significant for survival (OS p = 0.006, DFS p < 0.001). The PRSC differentiated between three prognostic groups (OS and DFS p < 0.001), and was superior compared to the stratification using MPR alone or the TNM8 systems, visualized by lower Akaike (AIC) and Bayesian information criterion (BIC) values. In the multivariate analyses, stage III tumors (HR 4.956, p = 0.003), tumors without MPR (HR 2.432, p = 0.015), and PRSC high-risk tumors (HR 5.692, p < 0.001) had significantly increased risks of occurring death. In conclusion, we support 65% as the optimal cut-off for MPR in adenocarcinomas. TNM8 and MPR were comparable regarding their prognostic significance. The novel prognostic score performed distinctly better regarding OS and DFS.

CD70 reverse signaling enhances NK cell function and immunosurveillance in CD27-expressing B-cell malignancies.

The interaction of the tumor necrosis factor receptor (TNFR) CD27 with its ligand CD70 is an emerging target to treat cancer. CD27 signaling provides costimulatory signals to cytotoxic T cells but also increases the frequency of regulatory T cells. Similar to other TNFR ligands, CD70 has been shown to initiate intracellular signaling pathways (CD70 reverse signaling). CD27 is expressed on a majority of B-cell non-Hodgkin lymphoma, but its role in the immune control of lymphoma and leukemia is unknown. We therefore generated a cytoplasmic deletion mutant of CD27 (CD27-trunc) to study the role of CD70 reverse signaling in the immunosurveillance of B-cell malignancies in vivo. Expression of CD27-trunc on malignant cells increased the number of tumor-infiltrating interferon γ-producing natural killer (NK) cells. In contrast, the antitumoral T-cell response remained largely unchanged. CD70 reverse signaling in NK cells was mediated via the AKT signaling pathway and increased NK cell survival and effector function. The improved immune control by activated NK cells prolonged survival of CD27-trunc-expressing lymphoma-bearing mice. Finally, CD70 reverse signaling enhanced survival and effector function of human NK cells in a B-cell acute lymphoblastic leukemia xenotransplants model. Therefore, CD70 reverse signaling in NK cells contributes to the immune control of CD27-expressing B-cell lymphoma and leukemia.

Induction chemoradiation in stage IIIA/N2 non-small-cell lung cancer: a phase 3 randomised trial.

BACKGROUND: One of the standard options in the treatment of stage IIIA/N2 non-small-cell lung cancer is neoadjuvant chemotherapy and surgery. We did a randomised trial to investigate whether the addition of neoadjuvant radiotherapy improves outcomes. METHODS: We enrolled patients in 23 centres in Switzerland, Germany and Serbia. Eligible patients had pathologically proven, stage IIIA/N2 non-small-cell lung cancer and were randomly assigned to treatment groups in a 1:1 ratio. Those in the chemoradiotherapy group received three cycles of neoadjuvant chemotherapy (100 mg/m(2) cisplatin and 85 mg/m(2) docetaxel) followed by radiotherapy with 44 Gy in 22 fractions over 3 weeks, and those in the control group received neoadjuvant chemotherapy alone. All patients were scheduled to undergo surgery. Randomisation was stratified by centre, mediastinal bulk (less than 5 cm vs 5 cm or more), and weight loss (5% or more vs less than 5% in the previous 6 months). The primary endpoint was event-free survival. Analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00030771. FINDINGS: From 2001 to 2012, 232 patients were enrolled, of whom 117 were allocated to the chemoradiotherapy group and 115 to the chemotherapy group. Median event-free survival was similar in the two groups at 12·8 months (95% CI 9·7-22·9) in the chemoradiotherapy group and 11·6 months (8·4-15·2) in the chemotherapy group (p=0·67). Median overall survival was 37·1 months (95% CI 22·6-50·0) with radiotherapy, compared with 26·2 months (19·9-52·1) in the control group. Chemotherapy-related toxic effects were reported in most patients, but 91% of patients completed three cycles of chemotherapy. Radiotherapy-induced grade 3 dysphagia was seen in seven (7%) patients. Three patients died in the control group within 30 days after surgery. INTERPRETATION: Radiotherapy did not add any benefit to induction chemotherapy followed by surgery. We suggest that one definitive local treatment modality combined with neoadjuvant chemotherapy is adequate to treat resectable stage IIIA/N2 non-small-cell lung cancer. FUNDING: Swiss State Secretariat for Education, Research and Innovation (SERI), Swiss Cancer League, and Sanofi.

TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

Microbiota-derived compounds drive steady-state granulopoiesis via MyD88/TICAM signaling.

Neutropenia is probably the strongest known predisposition to infection with otherwise harmless environmental or microbiota-derived species. Because initial swarming of neutrophils at the site of infection occurs within minutes, rather than the hours required to induce "emergency granulopoiesis," the relevance of having high numbers of these cells available at any one time is obvious. We observed that germ-free (GF) animals show delayed clearance of an apathogenic bacterium after systemic challenge. In this article, we show that the size of the bone marrow myeloid cell pool correlates strongly with the complexity of the intestinal microbiota. The effect of colonization can be recapitulated by transferring sterile heat-treated serum from colonized mice into GF wild-type mice. TLR signaling was essential for microbiota-driven myelopoiesis, as microbiota colonization or transferring serum from colonized animals had no effect in GF MyD88(-/-)TICAM1(-/-) mice. Amplification of myelopoiesis occurred in the absence of microbiota-specific IgG production. Thus, very low concentrations of microbial Ags and TLR ligands, well below the threshold required for induction of adaptive immunity, sets the bone marrow myeloid cell pool size. Coevolution of mammals with their microbiota has probably led to a reliance on microbiota-derived signals to provide tonic stimulation to the systemic innate immune system and to maintain vigilance to infection. This suggests that microbiota changes observed in dysbiosis, obesity, or antibiotic therapy may affect the cross talk between hematopoiesis and the microbiota, potentially exacerbating inflammatory or infectious states in the host.

Neoadjuvant chemotherapy and extrapleural pneumonectomy of malignant pleural mesothelioma with or without hemithoracic radiotherapy (SAKK 17/04): a randomised, international, multicentre phase 2 trial.

BACKGROUND: Postoperative hemithoracic radiotherapy has been used to treat malignant pleural mesothelioma, but it has not been assessed in a randomised trial. We assessed high-dose hemithoracic radiotherapy after neoadjuvant chemotherapy and extrapleural pneumonectomy in patients with malignant pleural mesothelioma. METHODS: We did this phase 2 trial in two parts at 14 hospitals in Switzerland, Belgium, and Germany. We enrolled patients with pathologically confirmed malignant pleural mesothelioma; resectable TNM stages T1-3 N0-2, M0; WHO performance status 0-1; age 18-70 years. In part 1, patients were given three cycles of neoadjuvant chemotherapy (cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 given every 3 weeks) and extrapleural pneumonectomy; the primary endpoint was complete macroscopic resection (R0-1). In part 2, participants with complete macroscopic resection were randomly assigned (1:1) to receive high-dose radiotherapy or not. The target volume for radiotherapy encompassed the entire hemithorax, the thoracotomy channel, and mediastinal nodal stations if affected by the disease or violated surgically. A boost was given to areas at high risk for locoregional relapse. The allocation was stratified by centre, histology (sarcomatoid vs epithelioid or mixed), mediastinal lymph node involvement (N0-1 vs N2), and T stage (T1-2 vs T3). The primary endpoint of part 1 was the proportion of patients achieving complete macroscopic resection (R0 and R1). The primary endpoint in part 2 was locoregional relapse-free survival, analysed by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00334594. FINDINGS: We enrolled patients between Dec 7, 2005, and Oct 17, 2012. Overall, we analysed 151 patients receiving neoadjuvant chemotherapy, of whom 113 (75%) had extrapleural pneumonectomy. Median follow-up was 54·2 months (IQR 32-66). 52 (34%) of 151 patients achieved an objective response. The most common grade 3 or 4 toxic effects were neutropenia (21 [14%] of 151 patients), anaemia (11 [7%]), and nausea or vomiting (eight [5%]). 113 patients had extrapleural pneumonectomy, with complete macroscopic resection achieved in 96 (64%) of 151 patients. We enrolled 54 patients in part 2; 27 in each group. The main reasons for exclusion were patient refusal (n=20) and ineligibility (n=10). 25 of 27 patients completed radiotherapy. Median total radiotherapy dose was 55·9 Gy (IQR 46·8-56·0). Median locoregional relapse-free survival from surgery, was 7·6 months (95% CI 4·5-10·7) in the no radiotherapy group and 9·4 months (6·5-11·9) in the radiotherapy group. The most common grade 3 or higher toxic effects related to radiotherapy were nausea or vomiting (three [11%] of 27 patients), oesophagitis (two [7%]), and pneumonitis (two [7%]). One patient died of pneumonitis. We recorded no toxic effects data for the control group. INTERPRETATION: Our findings do not support the routine use of hemithoracic radiotherapy for malignant pleural mesothelioma after neoadjuvant chemotherapy and extrapleural pneumonectomy. FUNDING: Swiss Group for Clinical Cancer Research, Swiss State Secretariat for Education, Research and Innovation, Eli Lilly.

A retrospective study of 1- versus 2-cm excision margins for cutaneous malignant melanomas thicker than 2 mm.

BACKGROUND: Most guidelines recommend at least 2-cm excision margin for melanomas thicker than 2 mm. OBJECTIVE: We evaluated whether 1- or 2-cm excision margins for melanoma (>2 mm) result in different outcomes. METHODS: This is a retrospective cohort study on patients with melanomas (>2 mm) who underwent tumor excision with 1-cm (228 patients) or 2-cm (97 patients) margins to investigate presence of local recurrences, locoregional and distant metastases, and disease-free and overall survival. RESULTS: In all, 325 patients with mean age of 61.84 years and Breslow thickness of 4.36 mm were considered for the study with a median follow-up of 1852 days (1995-2012). There was no significant difference in the frequency of locoregional and distant metastasis between the 2 groups (P = .311 and .571). The survival analysis showed no differences for disease-free (P = .800; hazard ratio 0.948; 95% confidence interval 0.627-1.433) and overall (P = .951; hazard ratio 1.018; 95% confidence interval 0.575-1.803) survival. LIMITATIONS: The study was not prospectively randomized. CONCLUSIONS: Our study did not show any significant differences in important outcome parameters such as local or distant metastases and overall survival. A prospective study testing 1- versus 2-cm excision margin is warranted.

Immunologic response to the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors.

PURPOSE: Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses. EXPERIMENTAL DESIGN: This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide(®) ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 µg. Secondary objectives included safety, tolerability, and clinical efficacy. RESULTS: In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event. CONCLUSIONS: Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.

Cilengitide treatment of newly diagnosed glioblastoma patients does not alter patterns of progression.

The integrin antagonist cilengitide has been explored as an adjunct with anti-angiogenic properties to standard of care temozolomide chemoradiotherapy (TMZ/RT → TMZ) in newly diagnosed glioblastoma. Preclinical data as well as anecdotal clinical observations indicate that anti-angiogenic treatment may result in altered patterns of tumor progression. Using a standardized approach, we analyzed patterns of progression on MRI in 21 patients enrolled onto a phase 2 trial of cilengitide added to TMZ/RT → TMZ in newly diagnosed glioblastoma. Thirty patients from the experimental treatment arm of the EORTC/NCIC pivotal TMZ trial served as a reference. MRIcro software was used to map location and extent of initial preoperative and recurrent tumors on MRI of both groups into the same stereotaxic space which were then analyzed using an automated tool of image analysis. Clinical and outcome data of the cilengitide-treated patients were similar to those of the EORTC/NCIC trial except for a higher proportion of patients with a methylated O(6)-methylguanyl-DNA-methyltransferase gene promoter. Analysis of recurrence pattern revealed neither a difference in the size of the recurrent tumor nor in the distance of the recurrences from the preoperative tumor location between groups. Overall frequencies of distant recurrences were 20 % in the reference group and 19 % (4/21 patients) in the cilengitide group. Compared with TMZ/RT → TMZ alone, the addition of cilengitide does not alter patterns of progression. This analysis does not support concerns that integrin antagonism by cilengitide may induce a more aggressive phenotype at progression, but also provides no evidence for an anti-invasive activity of cilengitide in patients with newly diagnosed glioblastoma.

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Molecular and immunological mechanisms of clonal evolution in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy characterized by the proliferation of clonal plasma cells in the bone marrow (BM). It is known that early genetic mutations in post-germinal center B/plasma cells are the cause of myelomagenesis. The acquisition of additional chromosomal abnormalities and distinct mutations further promote the outgrowth of malignant plasma cell populations that are resistant to conventional treatments, finally resulting in relapsed and therapy-refractory terminal stages of MM. In addition, myeloma cells are supported by autocrine signaling pathways and the tumor microenvironment (TME), which consists of diverse cell types such as stromal cells, immune cells, and components of the extracellular matrix. The TME provides essential signals and stimuli that induce proliferation and/or prevent apoptosis. In particular, the molecular pathways by which MM cells interact with the TME are crucial for the development of MM. To generate successful therapies and prevent MM recurrence, a thorough understanding of the molecular mechanisms that drive MM progression and therapy resistance is essential. In this review, we summarize key mechanisms that promote myelomagenesis and drive the clonal expansion in the course of MM progression such as autocrine signaling cascades, as well as direct and indirect interactions between the TME and malignant plasma cells. In addition, we highlight drug-resistance mechanisms and emerging therapies that are currently tested in clinical trials to overcome therapy-refractory MM stages.

IL-33-ST2 signaling promotes stemness in subtypes of myeloid leukemia cells through the Wnt and Notch pathways.

Cell stemness is characterized by quiescence, pluripotency, and long-term self-renewal capacity. Therapy-resistant leukemic stem cells (LSCs) are the primary cause of relapse in patients with chronic and acute myeloid leukemia (CML and AML). However, the same signaling pathways frequently support stemness in both LSCs and normal hematopoietic stem cells (HSCs), making LSCs difficult to therapeutically target. In cell lines and patient samples, we found that interleukin-33 (IL-33) signaling promoted stemness only in leukemia cells in a subtype-specific manner. The IL-33 receptor ST2 was abundant on the surfaces of CD34+ BCR/ABL1 CML and CD34+ AML cells harboring AML1/ETO and DEK/NUP214 translocations or deletion of chromosome 9q [del(9q)]. The cell surface abundance of ST2, which was lower or absent on other leukemia subtypes and HSCs, correlated with stemness, activated Wnt signaling, and repressed Notch signaling. IL-33-ST2 signaling promoted the maintenance and expansion of AML1/ETO-, DEK/NUP214-, and BCR/ABL1-positive LSCs in culture and in mice by activating Wnt, MAPK, and NF-κB signaling. Wnt signaling and its inhibition of the Notch pathway up-regulated the expression of the gene encoding ST2, thus forming a cell-autonomous loop. IL-33-ST2 signaling promoted the resistance of CML cells to the tyrosine kinase inhibitor (TKI) nilotinib and of AML cells to standard chemotherapy. Thus, inhibiting IL-33-ST2 signaling may target LSCs to overcome resistance to chemotherapy or TKIs in these subtypes of leukemia.

MEK1 drives oncogenic signaling and interacts with PARP1 for genomic and metabolic homeostasis in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a lethal malignancy etiologically caused by asbestos exposure, for which there are few effective treatment options. Although asbestos carcinogenesis is associated with reactive oxygen species (ROS), the bona fide oncogenic signaling pathways that regulate ROS homeostasis and bypass ROS-evoked apoptosis in MPM are poorly understood. In this study, we demonstrate that the mitogen-activated protein kinase (MAPK) pathway RAS-RAF-MEK-ERK is hyperactive and a molecular driver of MPM, independent of histological subtypes and genetic heterogeneity. Suppression of MAPK signaling by clinically approved MEK inhibitors (MEKi) elicits PARP1 to protect MPM cells from the cytotoxic effects of MAPK pathway blockage. Mechanistically, MEKi induces impairment of homologous recombination (HR) repair proficiency and mitochondrial metabolic activity, which is counterbalanced by pleiotropic PARP1. Consequently, the combination of MEK with PARP inhibitors enhances apoptotic cell death in vitro and in vivo that occurs through coordinated upregulation of cytotoxic ROS in MPM cells, suggesting a mechanism-based, readily translatable strategy to treat this daunting disease. Collectively, our studies uncover a previously unrecognized scenario that hyperactivation of the MAPK pathway is an essential feature of MPM and provide unprecedented evidence that MAPK signaling cooperates with PARP1 to homeostatically maintain ROS levels and escape ROS-mediated apoptosis.

Tumour mutations in long noncoding RNAs enhance cell fitness.

Long noncoding RNAs (lncRNAs) are linked to cancer via pathogenic changes in their expression levels. Yet, it remains unclear whether lncRNAs can also impact tumour cell fitness via function-altering somatic "driver" mutations. To search for such driver-lncRNAs, we here perform a genome-wide analysis of fitness-altering single nucleotide variants (SNVs) across a cohort of 2583 primary and 3527 metastatic tumours. The resulting 54 mutated and positively-selected lncRNAs are significantly enriched for previously-reported cancer genes and a range of clinical and genomic features. A number of these lncRNAs promote tumour cell proliferation when overexpressed in in vitro models. Our results also highlight a dense SNV hotspot in the widely-studied NEAT1 oncogene. To directly evaluate the functional significance of NEAT1 SNVs, we use in cellulo mutagenesis to introduce tumour-like mutations in the gene and observe a significant and reproducible increase in cell fitness, both in vitro and in a mouse model. Mechanistic studies reveal that SNVs remodel the NEAT1 ribonucleoprotein and boost subnuclear paraspeckles. In summary, this work demonstrates the utility of driver analysis for mapping cancer-promoting lncRNAs, and provides experimental evidence that somatic mutations can act through lncRNAs to enhance pathological cancer cell fitness.

Elimination of chronic viral infection by blocking CD27 signaling.

Neutralizing antibody (nAb) responses to lymphocytic choriomeningitis virus (LCMV) in mice and immunodeficiency virus and hepatitis C virus in humans are usually weak and slow to develop. This may be the result of structural properties of the surface glycoprotein, a low frequency of B cells with neutralizing specificity, and the necessity of prolonged affinity maturation of specific nAbs. In this study, we show that during LCMV infection, CD27 signaling on CD4+ T cells enhances the secretion of interferon-gamma and tumor necrosis factor-alpha. These inflammatory cytokines lead to the destruction of splenic architecture and immunodeficiency with reduced and delayed virus-specific nAb responses. Consequently, infection with the otherwise persistent LCMV strain Docile was eliminated after CD27 signaling was blocked. Our data provide a novel mechanism by which LCMV avoids nAb responses and suggest that blocking the CD27-CD70 interaction may be an attractive strategy to prevent chronic viral infection.