The prognosis for children with recurrent and/or refractory neuroblastoma (NB) is dismal. The receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is highly expressed on the surface of NB cells, provides a potential target for novel immunotherapeutics. Anti-ROR1 chimeric antigen receptor engineered ex vivo expanded peripheral blood natural killer (anti-ROR1 CAR exPBNK) cells represent this approach. N-803 is an IL-15 superagonist with enhanced biological activity. In this study, we investigated the in vitro and in vivo anti-tumor effects of anti-ROR1 CAR exPBNK cells with or without N-803 against ROR1(+) NB models. Compared to mock exPBNK cells, anti-ROR1 CAR exPBNK cells had significantly enhanced cytotoxicity against ROR1(+) NB cells, and N-803 further increased cytotoxicity. High-dimensional analysis revealed that N-803 enhanced Stat5 phosphorylation and Ki67 levels in both exPBNK and anti-ROR1 CAR exPBNK cells with or without NB cells. In vivo, anti-ROR1 CAR exPBNK plus N-803 significantly (p < 0.05) enhanced survival in human ROR1(+) NB xenografted NSG mice compared to anti-ROR1 CAR exPBNK alone. Our results provide the rationale for further development of anti-ROR1 CAR exPBNK cells plus N-803 as a novel combination immunotherapeutic for patients with recurrent and/or refractory ROR1(+) NB.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of circPCMTD1 in BCR/ABL1-positive myeloid leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR/ABL1 translocations. RNA sequencing and mass cytometry experiments identified aberrant activation of the DNA damage response (DDR) pathway as a downstream effect of circPCMTD1 depletion. DNA fiber assays, Comet assays and profiling of DDR markers (phospho-H2AX, phospho-CHK1, etc.) further underscored the pronounced effect of circPCMTD1 depletion in increasing genotoxic stress and inhibiting leukemic cell growth. circPCMTD1 targeting also led to aberrant DDR activation in leukemia patient blasts with BCR/ABL1 translocations. In in vivo experiments, circPCMTD1 knock-down prolonged the survival of mice engrafted with BCR/ABL1-positive leukemia cells. Mechanistically, we found that circPCMTD1 is enriched in the cytoplasm and associates with the ribosomes of leukemic blasts. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 generates a 127 amino-acid peptide product (cPCMTD1-127aa). Using a custom-produced antibody, we found that the cPCMTD1-127aa interacts with the BCR/ABL1 oncoprotein, as well as with the BLM, TOP3A and RMI1 proteins, which form the BTR complex and regulate DNA repair and genome stability. cPCMTD1-127aa enhanced BTR complex formation, thereby increasing cellular tolerance to genotoxic stress. In summary, we identify and characterize circPCMTD1 as a molecular vulnerability and potential therapeutic target in BCR/ABL1-positive leukemias.

Mass cytometers can record 40-50 parameters per single cell for millions of cells in a sample, and in particular, for leukemic cells. Many methods have been developed to cluster phenotypically similar cells within cytometry data, but there are fewer methods to visualize activity and interactions of pairs of proteins across these populations. We have developed a workflow for analyzing correlations associated with predfined populations. By clustering blood samples from acute myeloid leukemia (AML) patients and normal controls using an established algorithm, we obtained a minimum spanning tree of clusters of single cells. Using surface marker expression, we identified clusters on the tree that belonged to phenotypes of interest. Next, we computed correlations between pairs of proteins in each cluster. We developed a novel, coherent, probability-based statistic to test differences between vectors of correlation coefficients. By comparing all combinations of the normal controls under the statistic, we created an empirical distribution that provided a conservative measure of differential correlation. Using this empirically-derived distribution to define significance, we compared pooled samples from AML subtypes and normal controls to detect differential correlations. Given the structure present within this cytometry data set, we found it natural to consider correlations in this manner versus aggregating all data and computing a single correlation. Our results have the advantage that we can localize the statistical measure to determine contributions from particular phenotypic populations. Differentially correlated pairs of proteins can be further explored by considering a population’s distribution of correlation coefficients or biaxially plotting protein expressions within individual cells in a given population. Our approach leads to a better understanding of the nonlinear relationships that exist in the cytometry data. Author summary We introduce a novel method for analyzing the abundance of single cell data collected by high-throughput technologies. Due to the high dimensionality of such datasets, there is a need for methods to identify significant interactions between genes or proteins. In particular, we are interested in statistical differences between correlations of proteins within populations of cells determined by traditional immunophenotyping techniques. In this paper, we have demonstrated the utility of this new framework in the case of blood samples from individuals with different subtypes of leukemia and compare to healthy controls. The motivation for this application is that differences between can illuminate potential drivers of the disease. We have illustrated an example of how intracellular events can be detected by our statistic. Finally, this method is flexible in that it can also be applied in a variety of contexts where there are vectors of correlation coefficients that need to be compared.

Disease recurrence is the main cause of treatment failure after CD19-directed chimeric antigen receptor (CAR) T cells, often due to CD19 antigen loss, stability, and/or coverage. To overcome single-antigen escape, we evaluated a trispecific CAR targeting CD19, CD20, and CD22 with an OX40 costimulatory domain. Preclinical studies demonstrated potent, antigen-specific cytotoxicity in in vitro and in vivo lymphoma models. We then conducted a first-in-human phase I trial in patients with relapsed/refractory B-cell malignancies. Sixteen patients received infusions at a median vein-to-vein time of 7 days, at doses of 0.5 to 2 × 106 cells/kg. No severe cytokine release syndrome or neurotoxicity occurred. The overall response rate was 50%, including complete responses in 83% of patients with lymphoma. The 1-year overall survival rate was 61%, with durable remissions observed in lymphoma. CAR T expansion did not correlate with dose or response. T-cell exhaustion in apheresis cells correlated with progressive disease. Trispecific CAR T cells are safe and potentially active in lymphoma. SIGNIFICANCE:This first-in-human trial demonstrates that point-of-care manufactured trispecific CD19/20/22 CAR T cells with OX40 costimulation can be rapidly produced, safely administered, and induce durable remissions in relapsed B-cell lymphoma. Findings highlight multispecific targeting to mitigate antigen escape and identify T-cell exhaustion as a key biomarker guiding future CAR T optimization. See related commentary by Merz and Luttwak, p. 652.

Osteosarcoma (OSA) has a dismal prognosis despite surgical resection and multiagent chemotherapy. While adoptive natural killer (NK) cell therapies have been successful in hematological malignancies, the application in solid tumors is challenging due to a tumor microenvironment (TME) that impairs NK cell tumor infiltration. Here, we found that ex vivo expansion of NK cells significantly increases the expression of C-X-C motif chemokine receptor 3 (CXCR3), one of the major proteins in the regulation of NK cell chemotaxis. Engineered over-secretion of CXCR3 ligands, C-X-C motif chemokine ligand (CXCL)9, -10, or -11, from OSA cells significantly enhanced expanded NK cell migration toward OSA cells in vitro and infiltration into the TME in vivo, with the highest NK infiltration rate in CXCL10-secreting tumors. Infusions of expanded NK cells significantly reduced (p = 0.02), and concomitant treatment with an interleukin (IL)-15 agonist NKTR-255 further reduced tumor burden and significantly increased survival in mice bearing CXCL10-secreting tumors compared with those with wild-type tumors (p = 0.02). Single-cell RNA sequencing and mass cytometry revealed upregulated apoptosis and transforming growth factor-β (TGF-β) signaling as the potential mechanisms of response/resistance to NK cell therapy in vivo. Our findings highlight potential application of chemokine-enhanced NK tumor infiltration in combination with an IL-15 agonist as a novel approach to effective treatment of OSA.

Receptor signaling in Natural Killer (NK) cells leads to post-translational modification (e.g., phosphorylation) of sub-cellular signaling proteins within minutes of receptor stimulation that eventually give rise to diverse effector functions including cell proliferation. Recent single-cell mass cytometry (i.e., CyTOF) experiments in macrophages showed variations of abundances of phosphorylated signaling proteins across cell cycle states indicating a dependence of cell signaling kinetics on an order of magnitude slower kinetics (~ several hours) of cell cycle transitions. We investigated cell cycle dependence of NKG2D signaling kinetics in NK cells by CyTOF measurements performed on IL-2-treated NKG2D-stimulated primary human CD56dim NK cells. The CyTOF experiments revealed monotonic or semi-monotonic increases of the average protein abundances of the majority of signaling proteins such as pCrkL, pPLCγ2, and pErk, and the degranulation marker protein CD107a with progressing cell cycle states at specific time points post-NKG2D stimulation; however, several proteins such as pVav1, pS6, and pAkt, and early activation marker protein CD69 also showed non-monotonic variations in the average abundances with progressing cell cycle states. We used minimal mathematical and computational models coupling signaling and cell cycle processes to show that non-monotonic variations in the signaling protein abundances with progressing cell cycle stages are likely to arise in situations where protein synthesis and degradation and signaling kinetics are actively regulated by cell cycle processes. ### Competing Interest Statement The authors have declared no competing interest.

IL-12 is a potent NK cell-stimulating cytokine, but the presence of immunosuppressive myeloid cells such as myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK-cell cytotoxicity. Thus, we hypothesized that trabectedin, a myeloid cell-depleting agent, would improve the efficacy of IL-12 in triple-negative breast cancer (TNBC). In vitro treatment of healthy donor NK cells with trabectedin increased expression of the activation marker CD69 and mRNA expression of T-box transcription factor (Tbx21), the cytotoxic ligands TNF-related apoptosis-inducing ligand (TNFSF10), Fas ligand (FASLG), and the dendritic cell (DC)-recruiting chemokine lymphotactin (XCL1). The combination of IL-12 and trabectedin increased NK-cell cytotoxicity and activation and production of IFN-γ, TNF-α, and granzyme B in the presence of human TNBC cells. Treatment of 4T1 and EMT6 tumor-bearing mice with IL-12 and trabectedin led to a significant reduction in tumor burden compared with single-agent controls and the highest levels of plasma IFN-γ, intratumoral CD8+ T cells, and conventional type 1 DC. MDSC and M2-like macrophages were significantly decreased with combination therapy. NK-cell depletion abrogated the effects of combination therapy, as did the elimination of CD8+ T cells. NK-cell depletion led to lower levels of the NK cell-derived chemokine CCL5 and the DC-derived chemokine CXCL10, higher tumor burden, and decreased intratumoral CD8+ T cells. IL-12 and trabectedin also significantly enhanced the response of TNBC to anti-PD-L1 therapy. These data suggest that MDSC depletion augments the ability of IL-12-activated NK cells to drive the infiltration of DC and CD8+ T cells into TNBC for an antitumor effect.

This phase 1 study investigated the addition of gemtuzumab ozogamicin (GO) to intensive chemotherapy with cytarabine, daunorubicin, and midostaurin in 21 patients with newly diagnosed (ND) FMS-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML). Four dose levels of GO were evaluated. The use of GO was tolerable, with all dose-limiting toxicities similar to those seen in standard-of-care treatment. After induction, the median time to platelet recovery was 26 days, and the median time to absolute neutrophil count (ANC) recovery was 27 days. The maximum tolerated dose was cytarabine 100 mg/m2 on days 1 to 7, midostaurin 50 mg twice daily on days 8 to 21, daunorubicin 60 mg/m2 on days 1 to 3, and GO 3 mg/m2 on days 1 and 4. For the 18 patients who were evaluable for response after induction therapy, 16 patients (76%) achieved a composite complete response (complete remission [CR] + CR with incomplete hematologic recovery), and 2 (10%) had stable disease. Of the 14 patients who proceeded to consolidation, 5 discontinued the study for transplant, 1 for disease progression, and 1 for physician discretion. Seven patients completed consolidation therapy, all of whom achieved a CR. In total, 13 of the 21 patients (62%) received a hematopoietic stem cell transplant. Our results show that GO can safely be combined with intensive chemotherapy with midostaurin in ND, FLT3-mutated AML. This trial was registered at www.clinicaltrials.gov as #NCT03900949.

Venetoclax creates ongoing challenges when combined with posaconazole due to a known drug-drug interaction. Herein, we investigated the safety between venetoclax 100mg and 70mg when administered with posaconazole in acute myeloid leukemia in this single-center, retrospective comparative analysis. Primary safety endpoints were incidence/duration of cytopenias and incidence of tumor lysis syndrome during the first treatment cycle. A total of 113 patients received venetoclax 100mg while 32 patients received 70mg. Comparing venetoclax 100mg vs 70mg, no statistically significant differences were seen in grade 3 neutropenia (89.4% vs 84.4%, p=0.53), grade 4 neutropenia (88.5% vs 87.5%, p=1.0), median duration in days of grade 4 neutropenia (23 [range 1–105] vs 28 [range 1-81], p=0.35), grade 3/4 anemia (88.5% vs 84.4%, p=0.55), grade 3/4 thrombocytopenia (81.4% vs 87.5%, p=0.42), or tumor lysis syndrome (2.7% vs 6.3%, p=0.30). In adult patients with acute myeloid leukemia, a target dose of venetoclax 100mg with posaconazole may be a safe alternative. Further studies assessing dose optimization are warranted.

Abstract Low-grade gliomas (LGG) are the most common brain tumor in adolescents and young adults. LGG are neglected in the field of research due to their low relative risk compared to high-grade glioblastoma despite their potential to malignantly transform into fatal high-grade gliomas (HGG). Therefore, there is a necessity to investigate and develop novel therapeutics to combat the progression of glioma. Malignant progression of glioma is associated with an accumulation of immunosuppressive myeloid cells that impair anti-tumor T cell function. Trabectedin is an FDA approved chemotherapy used in the treatment of soft tissue sarcoma. Beyond its chemotherapeutic properties, Trabectedin has been shown to selectively deplete monocytes and macrophages, but its role in the modulating the brain TME has not yet been investigated. Here, we utilized mass cytometry (CyTOF) to investigate the impact of Trabectedin on the glioma immune TME as well as the global immune system in the immunocompetent, transgenic RCAS/t-va murine glioma model which recapitulates the progression of low to high-grade glioma. Trabectedin significantly reduced bone-marrow derived myeloid cells in the glioma TME as well as splenic macrophages and monocytes. Further, Trabectedin increased the proportion of tumor-infiltrating T cells. In conclusion, Trabectedin immunomodulates the glioma TME in vivo and could potentially enhance the efficacy of immunotherapies by overcoming myeloid-driven immunosuppressive resistance.

Introduction AML-MR is defined by cytogenetic and/or molecular abnormalities associated with myelodysplastic neoplasms or arising from a known history of myelodysplastic/ myeloproliferative neoplasms. T-AML is characterized by prior cytotoxic, radiation, or immunosuppressive therapy for an unrelated disease. Pts with AML-MR and t-AML have worse outcomes historically (Granfeldt Ostgard et al. J Clin Oncol 2015;33:3641). In the VIALE-A trial, only about 25% of pts had secondary AML (DiNardo et al. N Engl J Med 2020;383:617). In the VIALE-C trial, pts with secondary AML (38%) had worse survival than those with de novo AML (Wei et al. Blood 2020;135:2137). Both studies excluded pts with prior hypomethylating agent (HMA) exposure for prior MDS/MPN. Methods This study was a single-center, retrospective analysis of pts with AML-MR or t-AML, defined according to WHO 2022 classification (Khoury et al. Leukemia 2022;36:1703), who were treated with a venetoclax-based regimen from 10/2016 to 5/2023. Clinical, cytogenetic and molecular data were collected to establish diagnosis of AML-MR and t-AML. CRh, CRi, and CR were determined based on the 2022 ELN guidelines. Measurable residual disease (MRD) status measured by multiparameter flow cytometry was collected after cycles 2 and 3, with an MRD-positive threshold of ≥0.1%. Overall survival (OS) was calculated from time of venetoclax initiation. Early mortality was defined as death within 30 days after diagnosis. Kaplan-Meier method and the log-rank test were used to assess survival differences between AML-MR vs non-AML-MR and t-AML vs non-t-AML pts. Results Ninety-five pts who received venetoclax-based therapy were included. Median age was 67 years (range, 23-92), and 40 (42%) were female. Sixty-nine pts (73%) had AML-MR and 22 pts (23%) had t-AML. Fifteen pts had both AML-MR and t-AML. There was no significant difference in age, sex, or ECOG performance status between pts with AML-MR and those with non-AML-MR. Venetoclax first line was received by 30% of pts with AML-MR and 35% of those with non-AML-MR. Venetoclax was combined with HMA in 88% of pts with AML-MR and 96% of pts with non-AML-MR. The AML-MR group completed a median of 2 cycles (range, 1-14) and the non-AML-MR group completed a median of 4 cycles (range, 1-56) of venetoclax-based therapy. There were no significant differences in CRh, CRi, and CR rates between AML-MR vs non-AML-MR (composite CR/CRh/CRi 35% vs 46%). Among 25 pts with AML-MR who had MRD assessment at cycles 2 or 3, 12 (48%) were MRD-positive and 13 (52%) MRD-negative, whereas among 15 pts with non-AML-MR, 6 (40%) were MRD-positive and 9 (60%) MRD-negative. After excluding pts post-allogeneic transplant, there was no significant difference in the number of pts who proceeded to transplant [11/18 (61%) for AML-MR v. 54/70 (77%) for non-AML-MR, p = .23]. There was no significant difference in OS (median, 35.1 vs 41.3 months, p=0.85). Three pts with AML-MR and 0 with non-AML-MR suffered early mortality (p=0.56). There was no significant difference in age, sex or ECOG performance status between pts with t-AML and those with non-t-AML. Fifty percent of pts with t-AML and 33% with non-t-AML received venetoclax first line. Venetoclax with HMA was administered to 91% of pts with t-AML and 90% with non-t-AML. Pts with t-AML completed a median of 2 cycles (range, 1-13) and those with non-t-AML completed a median of 3 cycles (range, 1-56) of venetoclax-based therapy. There was no significant difference in CRh, CRi, and CR rate between t-AML vs non-t-AML (composite CR/CRh/CRi 27% vs 41%). Of the pts with t-AML who had MRD assessment at cycles 2 or 3, 5 (56%) were MRD-positive and 4 (44%) MRD-negative. Among pts with non-t-AML, 13 (42%) were MRD-positive and 18 (58%) were MRD-negative. After excluding pts post-allogeneic transplant, there was no significant difference in the number of pts who proceeded to transplant [4/22 (18%) with t-AML v. 14/66 (21%) with non-t-AML, p=1.00]. There was no significant difference in OS (median, 41.3 vs 35.1 months, p=0.74). One pt with t-AML and 2 pts with non-t-AML suffered early mortality. Conclusions In our real-world cohort, pts with AML-MR and those with t-AML treated with venetoclax had no significant differences in outcomes (CR, OS, and early death) compared with pts with non-AML-MR and non-t-AML, respectively. This data supports further investigation into the treatment of AML-MR and t-AML with venetoclax-based regimens.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p<0.001), as well as higher levels of IFN-γ (all p<0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p<0.01) and CXCL10 (p<0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p<0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

CD38 is a metabolically active enzyme broadly expressed on the surface of normal and malignant hematologic cells. It has been targeted clinically with anti-CD38 mAbs, for which efficacy may be limited by NK cell fratricide. Isatuximab is an anti-CD38 mAb that uniquely inhibits CD38 metabolic activity. Here, we used CRISPR/AAV to generate fratricide-resistant and metabolically-enhanced CD38KO/CD38-CAR NK cells using two isatuximab-based CD38 scFv (reversing heavy and light chain orientation) on the same CD8α/4-1BB/CD3ζ base, and we demonstrate their activity against a range of CD38-positive hematologic malignancies (acute myeloid leukemia, multiple myeloma, Burkitt lymphoma, and T-cell leukemia/lymphoma). The cytotoxicity of the CAR-NK cells was enhanced by upregulating CD38 expression on the malignant targets with all-trans retinoic acid (ATRA). By generating CD38KO/CD38-CAR T cells using the same engineering approach, we show that the CAR NK cells had higher cytotoxicity than CAR T cells against all hematologic tumor targets. Additionally, AAVS1KO/CD38-CAR NK cells were capable of targeting CD38 without experiencing fratricide and have a similar enhanced metabolic activity via the inhibitory activity of the cis-acting isatuximab-based scFv. Finally, we report fratricide-resistant CD38-CAR NK cells with enhanced metabolism and cytotoxicity toward CD38-positive hematologic malignancies, further increased by combination treatment with ATRA.

Circular RNAs (circRNAs) are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. CircRNAs are covalently joined and characterized by the perturbed arrangement of exons known as back-splicing. Initially regarded as transcriptional byproducts, circRNAs have been shown to regulate mRNA translation by acting as microRNA sponges, and recent studies have revealed their roles in transcription, translation, and various cellular functions. In cancer, circRNAs can function as oncogenes or tumor suppressors, and their stability makes them potential biomarkers for disease. In acute leukemias, circRNAs generated from recurrent chromosomal translocations contribute to leukemogenesis. Here, we investigate the role of circPCMTD1 in chronic myeloid leukemia (CML) in the blast crisis (BC). Functional studies using LNA-modified, RNase H-recruiting oligonucleotides (gapmers) targeting circPCMTD1 demonstrated a significant decrease in proliferation and a potent G2/M cell cycle blockade in CML-BC cell lines (K-562 & LAMA-84), both harboring the t(9;22)(q34;q11.2). Quantitative real-time PCR confirmed the specificity of circPCMTD1 depletion without affecting the linear PCMTD1 transcript. CircPCMTD1 knockdown (KD) reduced the viability of leukemic blasts, indicating its essential role in cell survival. RNA sequencing after circPCMTD1-KD in K-562 cells identified approximately 150 differentially expressed genes involved in cell cycle control, nuclear organization, and transcriptional regulation, such as SMARCA4, MACM, PCLAF, and SASH1. Gene Set Enrichment analysis highlighted rRNA processing and DNA replication-dependent chromatin function to be notably affected by circPCMTD1 depletion. CyTOF-based cell cycle analysis validated the G2/M blockade. Increased γH2AX levels indicated aberrant DNA damage response, confirmed by western blotting and intracellular flow cytometry. In addition, circPCMTD1-KD led to an increase in the phosphorylation of the CHK1, RPA32, ATR, ATM, and DNA-PK proteins. DNA fiber assays and comet assays further confirmed reduced DNA replication capacity and increased double-stranded DNA breaks upon circPCMTD1 depletion. Taken together, these data underscore the aberrant DNA damage response and the significant increase in genotoxic stress that is triggered by circPCMTD1 depletion. We performed targeted circPCMTD1 profiling in CML patients in the chronic, accelerated, and blast crisis phase and found an increased abundance of circPCMTD1 in advanced disease stages, indicating a potential role of higher circPCMTD1 expression in disease progression. In vitro experiments with patient blasts showed that circPCMTD1-KD increased γH2AX levels specifically in BCR::ABL-positive samples. In vivo, targeting circPCMTD1 in mice engrafted with BCR::ABL-positive blasts prolonged survival significantly, with no notable toxicities observed. Mechanistically, circPCMTD1 was enriched in the cytoplasm and associated with ribosomes. Polysome profiling suggested its protein-coding capacity, and we identified a cryptic open reading frame within circPCMTD1. Using custom antibodies, we detected a circPCMTD1-derived peptide (~30 KD) localized mainly in the nucleus. Immunoprecipitation followed by mass spectrometry revealed that the peptide interacted with BLM, TOP3A, and RMI1 proteins of the BTR complex. CircPCMTD1 knockdown reduced BTR complex formation. Knockdown of these proteins individually reduced leukemic blast viability, but concomitant depletion mimicked the G2/M blockade seen with circPCMTD1 depletion. Furthermore, treatment with Dasatinib, a tyrosine kinase inhibitor, decreased circPCMTD1-derived peptide levels without affecting the expression levels of the circPCMTD1 transcript and reduced BTR complex formation, linking BCR::ABL activity to circPCMTD1 function. In summary, we identify circPCMTD1 as a crucial regulator in BCR::ABL-positive leukemias, affecting DNA damage response, proliferation, and cell cycle progression. Our findings highlight circPCMTD1 as a potential therapeutic target in myeloid malignancies with t(9;22). Future studies should explore the therapeutic implications of targeting circPCMTD1 in combination with existing treatments, potentially offering a novel approach to managing drug resistance and improving outcomes in CML patients.

Low-grade glioma (LGG) is the most common brain tumor affecting pediatric patients (pLGG) and BRAF mutations constitute the most frequent genetic alterations. Within the spectrum of pLGGs, approximately 70%-80% - 80% of pediatric patients diagnosed with transforming pleomorphic xanthoastrocytoma (PXA) harbor the BRAF V600E mutation. However, the impact of glioma BRAF V600E cell regulation of tumor infiltrating fi ltrating immune cells and their contribution to tumor progression remains unclear. Moreover, the efficacy fi cacy of BRAF inhibitors in treating pLGGs is limited compared with their impact on BRAF-mutated melanoma. Here we report a novel immunocompetent RCAS-BRAF V600E murine glioma model. Pathological assessment indicates this model seems to be consistent with diffuse gliomas and morphological features of PXA. Our investigations revealed distinct immune cell signatures associated with increased trafficking fi cking and activation within the tumor microenvironment (TME). Intriguingly, immune system activation within the TME also generated pronounced inflammatory fl ammatory response associated with dysfunctional CD8+ + T cells, increased presence of immunosuppressive myeloid cells and regulatory T cells. Further, our data suggests tumor-induced inflammatory fl ammatory processes, such as cytokine storm. These fi ndings suggest a complex interplay between tumor progression and the robust inflammatory fl ammatory response within the TME in preclinical BRAF V600E LGGs, which may significantly influence fl uence animal survival.

Natural killer (NK)-cells have potent anti-tumor effects, yet it remains unclear if they are effective for patients with relapsed acute myeloid leukemia (AML). In a phase I clinical trial, we treated 12 patients (median age 60 years) with refractory AML (median 5 lines of prior therapy, median bone marrow blast count of 47%) with fludarabine/cytarabine followed by 6 infusions of NK-cells expanded from haploidentical donors using K562 feeder cells expressing membrane-bound IL21 and 4-1BBL. Patients received 106-107/kg/dose. No toxicity or graft-versus-host disease (GVHD) was observed and MTD was not reached. Seven patients (58.3%) responded and achieved a complete remission (CR) with/without count recovery. Median time to best response was 48 days. Five responding patients proceeded to a haploidentical transplant from the same donor. After a median follow-up of 52 months, 1-year overall survival (OS) for the entire group was 41.7%, better for patients who responded with CR/CRi (57.14%), and for patients who responded and underwent transplantation (60%). Persistence and expansion of donor-derived NK-cells were identified in patients' blood, and serum IFNγ levels rose concurrently with NK cell infusions. A higher count-functional inhibitory KIR was associated with higher likelihood of achieving CR/CRi. In conclusion, we observed a significant response to ex vivo expanded NK-cell administration in refractory AML patients without adverse effects.

Introduction: Approximately 25-30% of acute myeloid leukemia (AML) have FLT3 mutations, including internal tandem duplication (ITD) and tyrosine kinase domain mutations (TKD). Standard treatment for newly diagnosed (ND) FLT3 AML includes induction therapy with cytarabine and anthracycline and a FLT3 inhibitor such as midostaurin or quizartinib, followed by consolidation therapy +/- allogenic stem cell transplant (HSCT). FLT3 AML cells have significantly higher CD33 expression at diagnosis compared to non-FLT3 AML. Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to calicheamicin, a DNA damaging agent; currently approved for ND and relapsed/refractory CD33+ AML. Several studies have illustrated a benefit to adding GO to induction therapy, in patients with good and intermediate risk cytogenetics. Given the increased level of CD33 expression on blasts in FLT3 positive AML, we conducted an investigator-initiated study to assess safety and efficacy of addition of GO to induction therapy in ND FLT3 AML in combination with induction therapy with FLT3 inhibitor. Methods: In this phase I, open-label study (NCT03900949), eligible patients were 18 years or older with ND, CD33+, FLT3 AML (ITD or TKD). ECOG performance status 2 or less with adequate organ function was required. Patients with APL, isolated myeloid sarcoma, or CNS involvement were not eligible. Prior hydroxyurea was allowed; other systemic anti-AML therapies were prohibited. Four dose levels (DL) were assessed. Intravenous (IV) cytarabine (100mg/m2, days 1-7) and oral midostaurin (50mg bid, days 8-21) were the same across all DL. Daunorubicin (60mg/m2, days 1-3) was administered at DL1-3 , 90mg/m2, days 1-3 at DL4. GO was dosed at 3mg/m2 IV with increasing frequency as follows DL1: day 1; DL2: days 1 and 4; and DL3 and DL4: days 1, 4, and 7. Individual GO doses were limited to one 4.5 mg vial. Bayesian toxicity probability interval design “keyboard design” was used to guide dose escalations. Three patients were treated on DL1, 9 on DL 2, 6 on DL3, and 3 patients on DL4. Toxicities were summarized as the percent of patients who experienced the specific adverse event at the maximum grade. Overall survival (OS) was calculated from the start of treatment to death, censoring those alive at the last contact and analyzed using the Kaplan-Meier method. The study completed accrual in July 2024. The primary outcome was presence of dose-limiting toxicity (DLTs) during induction therapy. All patients were included in the efficacy and safety population for study analyses. Patients will be followed for up to two years after ending study treatment. Exploratory objectives included CD33 SNP and expression as it correlates to response and clinical outcomes. Results: Twenty-one patients with ND AML were enrolled. Median age was 51 years (range 20-73). Following induction therapy, 16 (76%) patients achieved a composite CR (CR+CRi), 2 (9.5%) had stable disease, 2 (9.5%) did not undergo disease assessments, and 1 patient has completed induction pending count recovery marrow . There were no DLTs observed on DL1 or DL4. One patient on DL2 experienced a hematologic DLT (inadequate count recovery by day 42 in the absence of disease). Two of 6 patients on DL3 experienced DLTs of GI toxicity resulting in less than 50% of the planned midostaurin administered. The most frequent toxicities overall were nausea (79%) and diarrhea (74%); the most frequent grade 3+ toxicities were white blood cell decreased (58%), and platelet count decreased (53%). DLT review of the final cohort will be available at time of presentation. Eleven patients received HSCT (52%), 7 (33%) have not, and 3 (14%) are being considered for transplant. Ten patients remain either on treatment or in study follow-up. Eleven patients have either completed study follow-up or died. OS was 79% at 6 months, 65% at 1 year, 52% at 1.5 years, and 39% at 2 years. Median survival follow-up was 367 days. Conclusions: The addition of CD33+ directed therapy, Gemtuzumab ozogamicin, to a standard of care cytarabine, anthracycline, and midostaurin induction regimen was found to be well-tolerated in ND, CD33+, FLT3-mutated AML patients, with toxicities similar to standard intensive induction therapy. The use of GO was tolerable as all DLTs were related to midostaurin and similar to those seen in standard of care treatment. The exploratory analyses are ongoing and will be reported at the main meeting.