ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma

IntroductionMetastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. MethodsHere, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and in a metastatic xenograft mouse model.ResultsOur results show that NK-92/5.28.z cells effectively kill RMS cells in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors.DiscussionThese findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS

Down syndrome and leukemia: from basic mechanisms to clinical advances

Children with Down syndrome (DS, trisomy 21) are at a significantly higher risk of developing acute leukemia compared to the overall population. Many studies investigating the link between trisomy 21 and leukemia initiation and progression have been conducted over the last two decades. Despite improved treatment regimens and significant progress in iden - tifying genes on chromosome 21 and the mechanisms by which they drive leukemogenesis, there is still much that is unknown. A focused group of scientists and clinicians with expertise in leukemia and DS met in October 2022 at the Jérôme Lejeune Foundation in Paris, France for the 1st International Symposium on Down Syndrome and Leukemia. This meeting was held to discuss the most recent advances in treatment regimens and the biology underlying the initiation, progression, and relapse of acute lymphoblastic leukemia and acute myeloid leukemia in children with DS. This review provides a summary of what is known in the field, challenges in the management of DS patients with leukemia, and key questions in the field

GATA1s induces hyperproliferation of eosinophil precursors in Down syndrome transient leukemia

Transient leukemia (TL) is evident in 5–10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1-mutations (GATA1s). Here we report that TL cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s-mutation as sorted TL-blasts, consistent with their clonal origin. TL-blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34+-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. While GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. ChIP-seq indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1Δe2 knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients

Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy

Histone deacetylase (HDAC)-inhibitors (HDACis) are well characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome associated myeloid leukemia (DS-AMKL) blasts. Investigating the anti-leukemic function of HDACis revealed their transcriptional and posttranslational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mTOR activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA-damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells

Combining LSD1 and JAK-STAT inhibition targets Down syndrome-associated myeloid leukemia at its core

Individuals with Down syndrome (DS) are predisposed to developing acute megakaryoblastic leukemia (ML-DS) within their first years of life [1]. Although, ML-DS is associated with a favorable prognosis, children with DS often experience severe toxicities from chemotherapy [2]. This highlights the unmet need for targeted therapies with improved risk profiles in this entity. Consequently, the aim of this study was to investigate a novel therapeutic approach specifically tailored to intervene with hallmarks of ML-DS leukemogenesis. The evolution of ML-DS occurs in a step-wise process originating from pre-malignant transient abnormal myelopoiesis (TAM) [3]. The molecular mechanisms underlying the progression from TAM to ML-DS are not fully understood. However, it was previously shown that epigenetic changes play a pivotal role in ML-DS leukemogenesis. The lysine demethylase LSD1 was identified as a crucial player in this process, as LSD1-driven gene signatures become activated in ML-DS [4]. Accordingly, RNA-sequencing analysis of pediatric acute myeloid leukemia (AML) subtypes revealed that LSD1 was highly expressed in acute megakaryoblastic leukemia (AMKL), and especially in TAM and ML-DS patients (Supplementary Fig. 1). LSD1 is essential for hematopoiesis, particularly during granulocytic and erythroid differentiation [5], and was shown to contribute to differentiation blockade in different AML subtypes [6,7,8]. Consequently, various irreversible LSD1 inhibitors have been developed, with some currently undergoing clinical trials for AML [9]. Therefore, we sought to investigate the rational use of LSD1 inhibitors in pediatric AMKL. The non-DS-AMKL cell line M-07e and the ML-DS cell line CMK were highly sensitive to irreversible LSD1 inhibition (IC50M-07e = 9.1 nM; IC50CMK = 38.8 nM; Supplementary Fig. 2A). Testing serial dilutions of the irreversible LSD1 inhibitor in non-DS-AMKL and ML-DS patient samples expanded via xenotransplantation (see Supplementary Table 1 for patient characteristics), both entities were equally sensitive to LSD1 inhibition (non-DS-AMKL: IC50#1 = 15.0 nM, IC50#2 = 2.0 nM; ML-DS: IC50#1 = 31.2 nM, IC50#2 = 17.1 nM, IC50#3 = 3.8 nM). All dose-response curves plateaued at a certain LSD1 inhibitor concentration (Supplementary Fig. 2B). The non-linear relationship between cytotoxicity and dosage points toward proliferation arrest and differentiation in response to LSD1 inhibition. In line with this, we observed myeloid differentiation upon visual inspection (Supplementary Fig. 3A) and upregulation of the myeloid markers CD86 and CD11b after 3 days of LSD1 inhibitor treatment (Supplementary Fig. 3B). These results revealed a potent proliferation block and induction of differentiation in non-DS-AMKL and ML-DS samples, however, the therapeutic efficacy of LSD1 inhibition may be limited by its non-linear dose-response relationship. Consequently, we aimed to design a rational drug combination to increase its anti-leukemic effects. Another hallmark of ML-DS development is the acquisition of activating mutations in Janus kinases (JAK) and cytokine receptors [4], promising potent anti-leukemic effects of the combination of LSD1 inhibition and the JAK1/JAK2 inhibitor ruxolitinib, as it was previously proposed for JAK2V617F mutated myeloproliferative neoplasms, secondary AML and a CSF3Rmut/CEBPαmut AML model [10,11,12]. Accordingly, pre-treatment with 350 nM LSD1 inhibitor for 3 days followed by exposure to serial dilutions of ruxolitinib led to synergistic growth inhibition in non-DS-AMKL and ML-DS cell lines (Supplementary Fig. 4), as well as in all ML-DS patient samples (Fig. 1A). The combination of LSD1 inhibition and ruxolitinib proved to be very effective in non-DS-AMKL blasts, however, with only additive cytotoxic effects in one of the two patient samples (Fig. 1A). Drug synergy in the ML-DS samples was confirmed when calculating the Bliss synergy scores (Fig. 1B). Interestingly, samples ML-DS #1 (JAK1mut) and #2 (wild-type for JAK1, JAK2, and JAK3, Supplementary Fig. 5) showed particularly high synergy scores (ML-DS #1 synergy score = 10.4; ML-DS #2 synergy score = 15.6; Fig. 1B). Contrary, the JAK3mut patient sample ML-DS #3 (Supplementary Fig. 5) only displayed mild drug synergy between LSD1 inhibition and ruxolitinib (synergy score = 2.0; Fig. 1B). Consequently, as ruxolitinib is a JAK1/JAK2 inhibitor, synergistic anti-leukemic effects seem to depend on JAK mutational status, which must be considered in future pre-clinical and clinical testing of this drug combination for ML-DS patients