Primary Leukemic Blasts Research Articles

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Gene Correction of DNMT3A:R882H in Primary Human AML Demonstrates That This Mutation Is Not Required for Disease Maintenance, but Is Associated with Increased Leukemia Stem Cell Frequency

Introduction: The genetic landscape of AML comprises numerous genetic alterations contributing to disease initiation and progression. Among these lesions, frequently occurring missense variants in DNMT3A:R882 are typically found as pre-leukemic events. An important and understudied question in leukemia pathogenesis is whether individual mutations are required for disease initiation, maintenance of established disease, or both. This is particularly the case with pre-leukemic mutations that affect the epigenome, as such mutations might be required to provide the epigenetic background necessary for disease initiation, but may no longer be required for disease maintenance. Here, we dissect the contribution of DNMT3A mutations to leukemic initiation and maintenance by gene correction in primary patient AML specimens. Methods: To functionally interrogate the contribution of DNMT3A mutations in human AML pathogenesis, we generated a novel CRISPR/Cas9-mediated toolset to selectively and specifically revert DNMT3A:R882 missense variants back to wildtype, or re-introduce the original mutation as a control, directly in both primary pre-leukemic HSCs (pHSCs) and primary leukemic blasts from 7 patient samples, leaving the remaining mutations and underlying germline background intact. Gene-corrected or re-mutated cells were subjected to functional assays both in vitro and in vivo. Results: We demonstrate that correction of DNMT3A mutations is feasible, achieving 5-40% gene editing both in purified pHSCs and leukemic blasts (n=7 patient samples). Using this approach, we first establish that DNMT3A mutations confer increased stemness in human pHSCs, with increasing serial replating capacity (mean increase re-mutated vs. corrected 5.53-fold, p<0.05). Surprisingly however, correction of DNMT3A mutations in leukemic blasts led to no immediate functional consequences. Specifically, primary patient leukemic blasts corrected for DNMT3A mutations retained the ability to engraft leukemia in NSGS mice and showed no difference in human chimerism or immunophenotype compared to re-mutated control from 5/5 primary patient samples. Importantly, limiting dilution transplantation assays (LDA) with two cases revealed no impact of DNMT3A mutations on leukemia-initiating cell (LIC) frequency in primary recipients, highlighting that DNMT3A mutations have no immediate effect on disease maintenance. We then performed whole-genome bisulfite sequencing and RNA-sequencing on leukemic blasts isolated from engrafted mice 4 months after DNMT3A correction or re-mutation. Global methylation levels between corrected and re-mutated cells were within the variance in either of these groups, and gene expression profiles were similarly driven by patient. However, paired analysis of each patient sample revealed that DNMT3A correction led to a consistent loss of stem cell expression signatures, suggesting influence of DNMT3A mutations on leukemia stem cell programs. Thus, we interrogated the effect of DNMT3A mutations on LSCs by performing secondary transplantations with two cases. In LDA assays, we observed a significant decrease in LIC frequency in the corrected leukemic blasts compared to re-mutated controls, indicating that correction of DNMT3A mutations induces a reduction in leukemia stem cell frequency, but does not completely abrogate leukemogenic potential. Conclusion: In summary, our work demonstrates a novel CRISPR/Cas9 approach to dissect the contribution of individual somatic lesions in human leukemia. The precise correction of single lesions while leaving co-occurring mutations intact allows us to perform controlled, functional genetic experiments on primary human AML. Here, we employ this approach to dissect the contribution of DNMT3A mutations to leukemia initiation and maintenance. We find that DNMT3A mutations impart increased stemness on human pHSCs, consistent with a key role in leukemia initiation. However, in primary patient blasts, we find that DNMT3A mutations are not required for leukemia maintenance. Strikingly, DNMT3A mutations in established leukemia were found to increase leukemia stem cell frequency. This approach will now allow for a detailed evaluation of differential methylation in mean and entropy and can be extended to other mutations of interest, extending our toolkit to unravel human AML biology directly in primary cancer specimens.

Preclinical evidence for the effective use of TL-895, a highly selective and potent second-generation BTK inhibitor, for the treatment of B-cell malignancies

TL-895 (formerly known as M7583) is a potent, highly selective, adenosine triphosphate (ATP)-competitive, second-generation, irreversible inhibitor of Bruton’s tyrosine kinase (BTK). We characterized its biochemical and cellular effects in in vitro and in vivo models. TL-895 was evaluated preclinically for potency against BTK using IC50 concentration–response curves; selectivity using a 270-kinase panel; BTK phosphorylation in Ramos Burkitt’s lymphoma cells by ProteinSimple Wes analysis of one study; anti-proliferative effects in primary chronic lymphocytic leukemia (CLL) blasts; cell viability effects in diffuse large B-cell lymphoma (DLBCL) and mantle-cell lymphoma (MCL) cell lines; effects on antibody-dependent cell-mediated cytotoxicity (ADCC) from Daudi cells and chromium-51 release from human tumor cell lines; and efficacy in vivo using four MCL xenograft model and 21 DLBCL patient-derived xenograft (PDX) models (subtypes: 9 ABC, 11 GCB, 1 Unclassified). TL-895 was active against recombinant BTK (average IC50 1.5 nM) and inhibited only three additional kinases with IC50 within tenfold of BTK activity. TL-895 inhibited BTK auto-phosphorylation at the Y223 phosphorylation site (IC50 1–10 nM). TL-895 inhibited the proliferation of primary CLL blasts in vitro and inhibited growth in a subset of activated DLBCL and MCL cell lines. TL-895 inhibited the ADCC mechanism of therapeutic antibodies only at supra-clinical exposure levels. TL-895 significantly inhibited tumor growth in the Mino MCL xenograft model and in 5/21 DLBCL PDX models relative to vehicle controls. These findings demonstrate the potency of TL-895 for BTK and its efficacy in models of B-cell lymphoma despite its refined selectivity.

SECTM1-based CAR T cells enriched with CD7-low/negative subsets exhibit efficacy in CD7-positive malignancies.

CD7 has been found to be a promising chimeric antigen receptor (CAR) T cell target in several clinical trials. However, its expression on normal T cells poses additional challenges in CD7-directed CAR therapy, such as complete fratricide, contamination with malignant cells, and immune suppression due to T-cell aplasia. By taking advantage of evolved affinity between ligand and receptor, we constructed a CD7-directed CAR with the extracellular domain of SECTM1, a natural ligand of CD7, as the recognition domain. SECTM1 CAR T cells killed the majority of T cells with high CD7 expression invitro. However, SECTM1 CAR Tcells with low or negative CD7 expression survived, expanded, and showed strong cytotoxicity to CD7+ malignant cell lines and primary leukemic blasts from patients with T-cell acute lymphoblastic leukemia and acute myelogenous leukemia invitro. It also exhibited efficacy in inhibiting xenograft tumor growth invivo. More exploration is needed for clinical efficacy potential to patients with CD7+ malignancies.

Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation.

Relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a significant challenge, with the re-emergence of the primary disease being the most frequent cause of death. Human leukocyte antigen (HLA)-DPB1 mismatch occurs in approximately 70% of unrelated allo-HSCT cases, and targeting mismatched HLA-DPB1 is considered reasonable for treating relapsed leukemia following allo-HSCT if performed under proper conditions. In this study, we established several clones restricted to HLA-DPB1*02:01, -DPB1*04:02, and -DPB1*09:01 from three patients who underwent HLA-DPB1 mismatched allo-HSCT using donor-derived alloreactive T cells primed to mismatched HLA-DPB1 in the recipient's body after transplantation. A detailed analysis of the DPB1*09:01-restricted clone 2A9 showed reactivity against various leukemia cell lines and primary myeloid leukemia blasts, even with low HLA-DP expression. T cell receptor (TCR)-T cells derived from clone 2A9 retained the ability to trigger HLA-DPB1*09:01-restricted recognition and lysis of various leukemia cell lines in vitro. Our study demonstrated that the induction of mismatched HLA-DPB1 specific T cell clones from physiologically primed post-allo-HSCT alloreactive CD4+ T cells and the redirection of T cells with cloned TCR cDNA by gene transfer are feasible as techniques for future adoptive immunotherapy.

Impact of rhG-CSF on Clinical Efficacy and Immune Cell Subsets after Initial Induction Chemotherapy in AML.

The impact of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in acute myeloid leukemia (AML) is still controversial. The purpose of this study is to explore the impact of rhG-CSF administration on clinical efficacy and immune cell subsets after initial induction chemotherapy in AML. The clinical efficacy and immune cell subsets were compared in the newly diagnosed patients with AML according to whether rhG-CSF was used after initial induction chemotherapy. Next, rhG-CSF stimulation experi-ments on leukemia cell lines and primary leukemia blasts were performed in vitro. There was no statistical difference between control group and rhG-CSF therapy group in complete remission rate and relapse free survival. The duration of agranulocytosis was significantly shortened in rhG-CSF therapy group compared with control group. The percentage of circulating monocytic myeloid-derived suppressor cells (M-MDSCs) and regulatory T cells (Tregs) were significantly increased after the administration of rhG-CSF. Furthermore, it was found that rhG-CSF did not promote the proliferation of leukemia cell lines and primary leukemia blasts, but increased the proportion of M-MDSCs and Tregs in vitro. Administration of rhG-CSF after initial induction therapy of AML does not affect the clinical remission and relapse rate, but reduces the duration of agranulocytosis and increases the proportion of M-MDSCs and Tregs.

Ex vivo Drug Sensitivity Imaging-based Platform for Primary Acute Lymphoblastic Leukemia Cells.

Resistance of acute lymphoblastic leukemia (ALL) cells to chemotherapy, whether present at diagnosis or acquired during treatment, is a major cause of treatment failure. Primary ALL cells are accessible for drug sensitivity testing at the time of new diagnosis or at relapse, but there are major limitations with current methods for determining drug sensitivity ex vivo. Here, we describe a functional precision medicine method using a fluorescence imaging platform to test drug sensitivity profiles of primary ALL cells. Leukemia cells are co-cultured with mesenchymal stromal cells and tested with a panel of 40 anti-leukemia drugs to determine individual patterns of drug resistance and sensitivity ("pharmacotype"). This imaging-based pharmacotyping assay addresses the limitations of prior ex vivo drug sensitivity methods by automating data analysis to produce high-throughput data while requiring fewer cells and significantly decreasing the labor-intensive time required to conduct the assay. The integration of drug sensitivity data with genomic profiling provides a basis for rational genomics-guided precision medicine. Key features Analysis of primary acute lymphoblastic leukemia (ALL) blasts obtained at diagnosis from bone marrow aspirate or peripheral blood. Experiments are performed ex vivo with mesenchymal stromal cell co-culture and require four days to complete. This fluorescence imaging-based protocol enhances previous ex vivo drug sensitivity assays and improves efficiency by requiring fewer primary cells while increasing the number of drugs tested to 40. It takes approximately 2-3 h for sample preparation and processing and a 1.5-hour imaging time. Graphical overview.

Abstract PR001: Mutant NPM1 hijacks transcriptional hub to maintain pathogenic gene programs and block differentiation in acute myeloid leukemia

Abstract Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of functions including ribosome biogenesis, mRNA processing, and maintenance of genomic stability. In acute myeloid leukemia (AML), the terminal exon of NPM1 is often mutated (~30% of adult AMLs), changing the nucleolar localization signal into a nuclear export signal, which results in a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have an aberrant expression of the HOXA genes, whose overexpression leads to leukemogenic transformation. The gene regulatory role of NPM1c has long been an enigma. Recently, it was shown that depletion or re-localization of the NPM1c protein into the nucleus [PG1] causes downregulation of the HOXA genes, leading to the speculation that NPM1c modulates transcription of HOXA genes. Here, we found NPM1c binds to chromatin and is located genome-wide at gene promoters and colocalized at active regions marked by H3K27ac in NPM1c leukemia cell lines and primary leukemia blasts. NPM1c clearly binds to known leukemia-driving genes - posterior HOXA, HOXB, and MEIS1/PBX3 genes as well as novel targets -IRX5 and NKX2-3. After PROTAC degradation of NPM1c, we observed a significant RNA polymerase II elongation defect genome-wide. The dramatic elongation defect is caused by the loss of the Super Elongation Complex (SEC) chromatin binding. Further tests suggest that loss of NPM1c leads to disruption of RNA polymerase II condensates as well as other direct transcriptional regulators of HOX genes - Menin and AFF4 through IDR interactions. We found that pulse PROTAC degradation of NPM1c leads to sustained differentiation with the loss of histone acetylation at NPM1c target loci. Further, NPM1c maintains the active chromatin state of target genes by antagonizing HDAC activity. Additionally, we found that NPM1c’s activation of the leukemia driving genes depends on the pre-established active chromatin state in the transformed hematopoietic stem and progenitor cells. NPM1c alone is not sufficient to activate these leukemia driving genes with heterochromatin marks accumulated. We also found that NPM1c binds to genomic regions that have been pre-bound by its export partner, XPO1. The XPO1 inhibitors Selinexor and Eltanexor can displace NPM1c from chromatin. The combination of clinical-grade XPO1 inhibitor eltanexor and Menin inhibitor MI-3454 displayed a strong synergy in inducing differentiation and reducing leukemia driving gene expression in the NPM1 mutated leukemia cell lines and PDX model, which promises to be an effective combination therapy for NPM1c leukemia patients. Citation Format: Xue Qing Wang, Dandan Fan, Yiman Liu, Qinyu Han, Jolanta Grembecka, Tomasz Cierpicki, Jianzhong Su, Shasha Chong, Liling Wan, Xiaotian Zhang. Mutant NPM1 hijacks transcriptional hub to maintain pathogenic gene programs and block differentiation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR001.

M2 Macrophages Drive Resistance to Phagocytosis and Improve Mitochondrial Metabolism in Acute Myeloid Leukemia Facilitating Leukemic Transformation and In Vivo Engraftment

Acute myeloid leukemia (AML) is a heterogeneous disease, which also extends to its tumor microenvironment (TME). Using single cell RNA-seq data (GSE116256), we performed differential gene expression analysis on macrophages from AML patients (AML-derived macrophages, AdMs) compared to healthy bone marrow macrophages. Thus, 306 upregulated genes in AdMs were identified as involved in different biological pathways such as proliferation, inflammation and cellular metabolism. Down-regulated genes (232) were mainly associated with the regulation of apoptosis. Single cell gene set enrichment analysis associated AdMs with an M2-like phenotype and an immunosuppression transcriptional program. We then investigated the macrophage landscape in a large cohort of AML patients by flow cytometry (n = 61) and immunohistochemistry, which revealed a large heterogeneity whereby AML patients with a predominant M2-like TME showed poor prognosis. Next, we evaluated the immune function of AdMs compared to healthy donor-derived macrophages. Thus, we showed that AdMs displayed decreased phagocytic capacity towards AML cells compared to healthy macrophages. Since AdMs might directly descend from the mutated leukemic clones we evaluated whether these mutations could affect the immune function of macrophages. Cord blood-derived CD34+ cells were lentivirally transduced with FLT3-ITD, differentiated towards macrophages, and these FLT3-ITD+ macrophages displayed decreased phagocytic activity. We questioned whether endogenous murine macrophages that are still present in immunodeficient mouse strains that are often used for human studies might form a barrier that needs to be taken to achieve efficient engraftment of human cells. Macrophages were isolated from NSG mice and were used in phagocytosis assays. The percentage of phagocytosed primary AML blasts by murine macrophages ranged between 3.2-51.4%, similar to phagocytosis levels observed when using human macrophages for the same AML samples. While performing these phagocytosis assays, we noted that the cells that remained and were not phagocytosed had an altered appearance and were very viable, as if these cells had gained fitness. We tested this further by exposing leukemic blasts to M2 macrophages for 48 h, after which the remaining cells were analyzed functionally. We observed that the 48 h "training" of primary leukemic blasts significantly enhanced their mitochondrial metabolism (in part mediated via mitochondrial transfer from macrophages to leukemic blasts), enhanced their in vitro and in vivo homing capacity and that they became resistant to phagocytosis. As a most striking example we used primary APL cells that are notoriously difficult to engraft in NSG mice, which we hypothesized could be in line with their high intrinsic susceptibility to macrophage-mediated phagocytosis. APL cells were exposed to M2 macrophages for 48h resulting in two cellular fates: 91.7% of cells were phagocytosed, but the remaining 8.3% APL blasts were able to induce fatal leukemia when transplanted into NSGS mice in all evaluated cases (n = 6), while prior to training these cells did not engraft long-term and were not able to confer disease. We also tested the tumor supportive capacity on M2 macrophages by performing intrafemoral injection in NSGS mice after which non-trained APL blasts were injected intravenously. Again, efficient engraftment and fatal leukemia development were observed, but only in mice co-injected with M2 macrophages. Strategies to target the supportive AdM subpopulation would be of interest. In our scRNA-seq analysis, we identified the metabolic enzyme Nicotinamide phosphoribosyltransferase (NAMPT) to be upregulated in AdMs. Ex vivo studies revealed that while conventional chemotherapeutics, AraC/venetoclax, efficiently target the AML blasts (CD34+/CD117+) with minimum effects on the AdMs, NAMPT inhibition with KPT-9274 was able to efficiently target both the AML blasts and the AdM fractions. Functionally, treatment of M2 macrophages with KPT-9274 shifted their polarization towards an M1 phenotype, with reduced support to AML cells in co-culture assays. In conclusion, we uncover the heterogeneity in the macrophage landscape in AML patients, show the functional relevance of M2-polarized macrophages for leukemic transformation, and provide alternative approaches for targeting aimed at the tumor-supportive microenvironment.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia

BackgroundNatural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK...

Conjoined Genes as Common Events in Childhood Acute Lymphoblastic Leukemia.

Simple SummaryAcute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. In recent years, broad application of NGS technologies enabled the discovery of novel genomically defined ALL. In this study, as a proof-of-principle, we applied RNA-seq technology to comprehensively profile the transcriptional landscape of a collection of 10 childhood BCP-ALL cases, and performed a deep bioinformatics analysis including several publicly available datasets, in order to characterize their full spectrum of transcriptional events. The paired-end RNA sequencing of our BCP-ALL pediatric cohort revealed a total of 9001 raw fusion events, which, after filtering, resulted in 245 candidate fusions. Overall, 235 out of 245 events were intra-chromosomal fusions, among which 229 involved two contiguous or overlapping genes, also known as conjoined genes (CGs). Among them, we identified a subset of 14 CGs (6.1%) exclusively expressed in leukemic cases but neither in solid cancers nor in normal samples. These events could be suggestive of a novel mechanism of transcriptional regulation in childhood leukemia and may represent novel potential leukemia-specific biomarkers.Acute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. For the last three decades, conventional cytogenetic and molecular approaches allowed the identification of genetic abnormalities having prognostic and therapeutic relevance. Although the current cure rate in pediatric B cell acute leukemia is approximately 90%, it remains one of the leading causes of mortality in childhood. Furthermore, in the contemporary protocols, chemotherapy intensity was raised to the maximal levels of tolerability, and further improvements in the outcome will depend on the characterization and reclassification of the disease, as well as on the development of new targeted drugs. The recent technological advances in genome-wide profiling techniques have allowed the exploration of the molecular heterogeneity of this disease, even though some potentially interesting biomarkers such as conjoined genes have not been deeply investigated yet. In the present study, we performed the transcriptome sequencing (RNA-seq) of 10 pediatric B cell precursor (BCP)-ALL cases with different risk (four standard- and six high-risk patients) enrolled in the Italian AIEOP-BFM ALL2000 protocol, in order to characterize the full spectrum of transcriptional events and to identify novel potential genetic mechanisms sustaining their different early response to therapy. Total RNA was extracted from primary leukemic blasts and RNA-seq was performed by Illumina technology. Bioinformatics analysis focused on fusion transcripts, originated from either inter- or intra-chromosomal structural rearrangements. Starting from a raw list of 9001 candidate events, by employing a custom-made bioinformatics pipeline, we obtained a short list of 245 candidate fusions. Among them, 10 events were compatible with chromosomal translocations. Strikingly, 235/245 events were intra-chromosomal fusions, 229 of which involved two contiguous or overlapping genes, resulting in the so-called conjoined genes (CGs). To explore the specificity of these events in leukemia, we performed an extensive bioinformatics meta-analysis and evaluated the presence of the fusions identified in our 10 BCP-ALL cohort in several other publicly available RNA-seq datasets, including leukemic, solid tumor and normal sample collections. Overall, 14/229 (6.1%) CGs were found to be exclusively expressed in leukemic cases, suggesting an association between CGs and leukemia. Moreover, CGs were found to be common events both in standard- and high-risk BCP-ALL patients and it might be suggestive of a novel potential transcriptional regulation mechanism active in leukemic cells.

Abstract 3001: Targeting autophagy in acute lymphoblastic leukemia: Synergism between ponatinib, hydroxychloroquine and rapamycin

Abstract Currently, the role of autophagy in acute lymphoblastic leukemia (ALL) is not fully understood however it has been linked to drug resistance. Several autophagy modulators have proven their efficacy in single agents or as chemo-sensitizer in ALL. Recently, the 3rd generation BCR-ABL1 inhibitor, ponatinib, has been showed to promote autophagy in chronic myeloid leukemia (CML) cells and the combination between ponatinib and hydroxychloroquine (HCQ) has proven its efficacy in neuroblastoma cells. Here, we tested the efficacy of combining different autophagy modulators (ponatinib, HCQ and rapamycin) against ALL cell lines and primary leukemic ALL cells. Firstly, we established the IC50 values of a panel of Philadelphia (Ph)-positive/negative B-/T-ALL cell lines (n=6) treated with ponatinib, HCQ and rapamycin in single agent. Then, using sub-toxic concentrations, we evaluated the efficacy of the combinations HCQ+Ponatinib and HCQ+Rapamycin in the reduction of the cell viability after 24, 48 and 72 hours of simultaneous treatment. Combination index analyses showed a synergic time and dosage-dependent reduction of the cell viability in all the cell lines treated with HCQ+Ponatinib. The combination HCQ+Rapamycin resulted synergic only in Ph-negative ALL cell lines. These results were confirmed by the significant induction of apoptosis (Annexin V and/or Caspase-3 staining) in the samples treated with HCQ+Ponatinib. The two combinations also significantly reduced the proliferation capacity of ALL cell lines in comparison with controls and single treatments. Moreover, the efficacy of the two combinations was confirmed on primary leukemic blasts isolated form adult ALL patients(n=6). Indeed, a significant induction of apoptosis was seen in samples treated with the two combinations in comparison with single treatments. To deeper understand the mechanism of action of the combinations we evaluated LC3A/B expression by flow cytometry. Interestingly, the expression of LC3A/B was modified heterogeneously among the different ALL cell lines and did not correlate with the response in term of cell viability or induction of apoptosis. Light microscopy analyses showed a significant increment in the number and diameter of autophagy vesicles in both ALL cell lines and primary leukemic ALL cells treated with HCQ+Ponatinib in comparison with single treatments. Interstingly, no significant effect on autophagy vesicles number or diameter was seen in the samples treated with HCQ+Rapamycin in comparison with single treatments. In conclusion, our findings show that the identified combinations effectively inhibit cell viability and induce apoptosis in Ph-positive/negative B-/T-ALL cells, suggesting the fundamental role of autophagy regulation in ALL survival. Citation Format: Andrea Ghelli Luserna Di Rora, Eugenio Fonzi, Lorenzo Ledda, Roberta Napolitano, Anna Ferrari, Antonella Padella, Martina Ghetti, Maria Teresa Bochicchio, Mouna Jandoubi, Cristina Mazzotti, Matteo Paganelli, Claudio Cerchione, Giovanni Marconi, Gerardo Musuraca, Giovanni Martinelli, Giorgia Simonetti. Targeting autophagy in acute lymphoblastic leukemia: Synergism between ponatinib, hydroxychloroquine and rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3001.

Integrated RNAi screening identifies the NEDDylation pathway as a synergistic partner of azacytidine in acute myeloid leukemia

Treatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

Reference Mapping Pediatric Leukemia Using Single Cell Multiomic Atlas of Pediatric Hematopoiesis

Acute lymphoblastic leukemia is the most common pediatric cancer and leading cause of cancer related mortality in pediatric populations. A key challenge to bridge better therapies to patients that fail conventional therapy are to understand their tumor landscape and aberrations in cell signaling, particularly in relation to normal hematopoietic development.To address this gap, we produced a unified reference map of pediatric T, B, and myeloid cell development from the HSPC using single cell RNA-seq and single-cell ATAC-seq on healthy pediatric bone marrow and thymus. We employed 6 different FACS sorting strategies in order to capture rare, but informative, progenitor cell states, including those of the CCR9+ CD34+ CD1A- CD4- CD8- early-T-cell precursor, CD34+ CD1A- CD4- CD8- pro-T cell, and CD34+ CD1A+ CD4- CD8- pre-T cell and Lin-CD34+CD38- multipotent, lymphoid, and myeloid progenitors from the bone marrow.We mapped leukemic cells from patients from 4 different subtypes of pediatric leukemia (T-ALL, ETP-ALL, MPAL, AML) to our healthy reference and found that our reference map can distinguish between subtle differences in transcriptome and epigenome that were undetectable using surface marker or canonical gene expression. Notably, using trajectories inferred from our healthy reference map, we discovered a large amount of inter-tumoral and intra-tumoral heterogeneity, with leukemic blasts from different patients and different populations within any one patient projecting to different cell states along normal development. Finally, we mapped engrafted leukemic cells from patient derived xenografts (PDX) back to our healthy reference. While we observed patient-specific transcriptomic shifts in engrafted versus primary leukemic blasts, we found that the overall transcriptomic hierarchy is maintained in the most PDX, with engrafted cells projecting to near-identical stages of arrest along our healthy hematopoietic trajectory. Interestingly, for PDX that projected to different areas in development compared to primary sample, we discovered alterations in expression of key transcription factors that regulate hematopoietic development.Our single cell multi-omic reference map of pediatric hematopoiesis serves as a valuable reference for mapping RNA-seq and ATAC-seq data back to nearest healthy precursors in normal hematopoietic development. On-going analysis is utilizing single cell transcriptomic, chromatin accessibility data from additional leukemic patients, including patients with B-ALL, to determined key genes and regulators that are altered in comparison to nearest healthy cell-types. In addition, population level signatures learned from healthy reference are being tested in bulk-transcriptomic ALL datasets. We are eager to present the results of these analyses at ASH.*CC and JX, as well as, DTT and KT contributed equally to this work [Display omitted] DisclosuresTeachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

Towards the Next Generation CAR T Cells with TEGs: In Vivo Efficacy — Toxicity Profile in Xenografts of Primary Human AML Disease and Healthy Bone Marrow

T.S. and I.J.G. contributed equally</spanBackgroundγδT cells mediate essential cancer immune surveillance by sensing transformation-dependent metabolic changes through their γδT cell receptors (TCRs). This process is independent ofMHC-restricted antigen presentation, making these innate-like receptors valuable additions to current treatment strategies. The γδTCR alone is instrumental to distinguish between healthy and leukemic stem cells. This concept led to the development of next generation CAR T cells, so-called TEGs: αβT cells Engineered to express a defined γδTCR. A particular γ9δ2TCR, isolated from “clone 5”, has been selected as the candidate for clinical testing (TEG001). A purification strategy was introduced that combined maximal interference of γδTCR chains with endogenous TCR chains with a GMP-grade anti-αβTCR-bead-based depletion strategy to obtain a pure population of engineered immune cells (Straetemans et al. Clin Can Res 2015). TEG001 cells showed a strong and broad recognition of hematological malignancies against both cell lines and primary AML in vitro and limited the tumor outgrowth in cell line-based mouse xenograft models (Marcu-Malina et al. Blood 2011, Gruender et al. Blood 2012). To further prepare clinical implementation of this novel strategy we here investigated the in vivo efficacy and toxicity profile against primary leukemic blasts and healthy hematopoietic cells, respectively.ResultsIn order to evaluate the biodistribution and safety profile of TEGs we developed patient derived xenograft (PD-X) in vivo models by establishing primary malignant AML blasts, which tested positive in an in vitro assay for recognition by TEGs, in NSG mice. In addition, healthy stem cells from human cord-blood were in parallel engrafted in a separate set of NSG mice. This allowed us to study the homing capacity, tumor-targeting efficacy and off-target toxicity of TEGs by following the outgrowth of malignant and healthy human cell fractions in the peripheral blood. Healthy hematological cellular compartments remained unharmed by TEGs, in spite of effector cell persistence for over 50 days. In contrast, AML blasts could be eliminated as measured by frequencies of blasts in peripheral blood.ConclusionWithin the limitations of humanized PD-X models, TEGs target acute myeloid leukemia but do neither interfere with engraftment of hematopoietic progenitors nor harm matured subsets of the hematopoiesis. In addition, no additional signs of off-target toxicity were observed in mice. TEGs are a promising addition to the currently available immune therapeutic strategies as they target cancer as a metabolic disorder. A GMP-compliant production procedure has now been established in order to evaluate TEG001 in a phase I open label dose escalation study to assess the safety of TEGs in patients with primary refractory or relapsed acute myeloid leukemia as well as patients with multiple myeloma. DisclosuresSebestyen:University Medical Center Utrecht: Patents & Royalties: inventor on the recognition mechanism of clone 5. Kuball:Gadeta (www.gadeta.nl): Consultancy, Equity Ownership, Patents & Royalties: on gdT cells and receptors and isolation strategies , Research Funding; Miltenyi: Research Funding.

M7583 Is a Highly Selective and Potent Second Generation BTK Inhibitor for Treatment of B-Cell Malignancies

▪The advent of ibrutinib has represented a new era in the treatment of B-cell malignancies, with remarkable clinical efficacy shown in previously difficult to treat lymphomas. Now, second generation Bruton's tyrosine kinase (BTK) inhibitors are in development with more refined pharmacologic profiles that maintain the potent anti-tumor effects of ibrutinib, but that improve upon overall tolerability and circumvent ibrutinib's inhibitory effect on antibody-dependent cell-mediated cytotoxicity (ADCC).M7583 is a potent, orally administered, highly selective, adenosine triphosphate (ATP)-competitive, second generation, irreversible inhibitor of BTK. Kinase specificity has been determined in vitro using the Merck Millipore Kinase Profiler panel. Only 3 kinases (Blk, BMX, and Txk) had IC50s within a 10-fold range of BTK (BTK IC50 = 18.5 nM; Blk IC50 = 77 nM; BMX IC50= 5 nM; Txk IC50 = 62 nM). Target inhibition was confirmed with a concentration dependent decrease in BTK auto-phosphorylation detected by western blot in the Ramos cell line (human Burkitt's B-cell lymphoma). To examine the impact of kinase selectivity on the overall efficacy of M7583, its preclinical activity was compared with that of ibrutinib in a series of in vitro studies. M7583 inhibited the growth of diffuse large B-cell lymphoma (DLBCL) cell lines and primary chronic lymphocytic leukemia (CLL) blasts. In vivo , treatment with M7583 inhibited tumor growth in mantle cell lymphoma cell line-derived xenografts and patient-derived xenograft (PDX) models of DLBCL with the activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) subtype being the most sensitive to M7583; consistent with the findings for other BTK inhibitors. To examine the potential for inhibition of ADCC by therapeutic antibodies, M7583 and ibrutinib were combined with rituximab or an ADCC incompetent version of rituximab in ADCC cell lysis experiments. At clinically relevant concentrations, ibrutinib inhibited the ADCC mechanism of rituximab, whereas M7583 did not. Collectively, these findings show the potency and selectivity of M7583 for BTK, as well as its efficacy in models of B-cell malignancies. The safety and efficacy of M7583 is currently being investigated in Phase I/II trials in patients with B-cell malignancies. DisclosuresGoodstal:EMD Serono: Employment. Ma:EMD Serono: Employment. Lin:EMD Serono: Employment. Crandall:EMD Serono: Employment. Crowley:EMD Serono: Employment. Bender:EMD Serono: Employment. Iadevaia:EMD Serono: Employment. Clark:EMD Serono: Employment.

A Cellular Antidote to Specifically Deplete Anti-CD19 Chimeric Antigen Receptor (CAR19) Positive Cells

Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19, CTL019) have led to impressive clinical responses in relapsing or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). With the likely imminent approval of CTL019 for patients with B-ALL, increasing numbers of patients will be exposed to this therapy. Last year we reported a rare case of a single patient who relapsed with CD19-negative, CAR19-expressing leukemia, likely due to inadvertent transduction of a leukemic cell with the CAR19 lentivirus during CTL019 manufacturing (Lacey SF, ASH, 2016 #281). The high expression of the CAR in leukemic cells and its absence from normal tissues make it an ideal engineered tumor target. We therefore developed anti-CAR19 CAR T cells with the goal of specifically targeting CAR19+ B-ALL.Methods. We designed two anti-CAR19 CAR using an anti-CAR19 idiotype scFv (clone 136.20.1, original clone kind gift of Dr. Laurence Cooper) testing two light chain orientations for the scFv: light to heavy (L2H) and heavy to light (H2L). We cloned the constructs into our standard backbone containing the CD8 hinge and trans-membrane domains, 4-1BB costimulatory and CD3z signaling domain. The constructs were packaged into a lentiviral vector and used to transduce normal donor T cells. As target cells, we used ex-vivo expanded primary leukemic blasts from the CAR19-expressing relapsed patient (CHP-107), as well as a luciferase+ B-ALL cell line (NALM6) transduced with the full-length CAR19 used in our clinical studies. Of note, the expression of CAR19 in CD19+ NALM6 cells led to apparent loss of CD19 by flow cytometry. We tested CART function in vitro with luciferase-based killing assays and in vivo in human xenograft models using NOD-SCID gamma chain deficient (NSG) mice.Results. Both L2H and H2L anti-CAR19 CAR were efficiently expressed on T cells as detected by flow cytometry. In vitro CART-CAR19 efficiently killed CAR19-expressing B-ALL (NALM6) (L2H orientation: p< 0.0001; H2L: p=0.0063) but not wild-type (WT) NALM6 (p=NS) (Figure 1 A). In vivo engraftment studies showed that expression of CAR19 did not impact the growth rate of NALM6. We then tested our lead anti-CAR19 CART (L2H) in vivo. NSG mice were engrafted with either CAR19+ NALM6 (CD19-) or luciferase-positive relapsed CHP107 leukemia blasts (CAR19+, CD19-). At day 7 or 14 mice were randomized to receive no treatment, control T cells (UTD), CART19 or CART-CAR19 L2H. In both xenograft models CART19 and UTD cells failed to control disease progression, whereas CART-CAR19 showed significant leukemia control (Figure 1 B).Conclusions. The acquisition of CAR expression by leukemic cells during CART manufacturing is a rare event that generates a tumor-specific antigen, thereby presenting an opportunity for specific targeting without off-target toxicity. In addition, we hypothesize that anti-CAR19 CART could be used as an “antidote” to CAR19 T cells for those patients who have been in stable deep remission for several years, in order to deplete their CAR T cells and alleviate B cell aplasia. [Display omitted] DisclosuresRuella:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding; Genentech: Honoraria. Melenhorst:Novartis: Research Funding. June:Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding; CARMA Therapeutics: Consultancy, Equity Ownership, Research Funding.

Targeting Lactate Metabolism by Inhibiting MCT1 or MCT4 Impairs Leukemic Cell Proliferation, Induces Two Different Related Death-Pathways and Increases Chemotherapeutic Sensitivity of Acute Myeloid Leukemia Cells.

Metabolism in acute myeloid leukemia (AML) cells is dependent primarily on oxidative phosphorylation. However, in order to sustain their high proliferation rate and metabolic demand, leukemic blasts use a number of metabolic strategies, including glycolytic metabolism. Understanding whether monocarboxylate transporters MCT1 and MCT4, which remove the excess of lactate produced by cancer cells, represent new hematological targets, and whether their respective inhibitors, AR-C155858 and syrosingopine, can be useful in leukemia therapy, may reveal a novel treatment strategy for patients with AML. We analyzed MCT1 and MCT4 expression and function in hematopoietic progenitor cells from healthy cord blood, in several leukemic cell lines and in primary leukemic blasts from patients with AML, and investigated the effects of AR-C155858 and syrosingopine, used alone or in combination with arabinosylcytosine, on leukemic cell proliferation. We found an inverse correlation between MCT1 and MCT4 expression levels in leukemic cells, and showed that MCT4 overexpression is associated with poor prognosis in AML patients. We also found that AR-C155858 and syrosingopine inhibit leukemic cell proliferation by activating two different cell-death related pathways, i.e., necrosis for AR-C155858 treatment and autophagy for syrosingopine, and showed that AR-C155858 and syrosingopine exert an anti-proliferative effect, additive to chemotherapy, by enhancing leukemic cells sensitivity to chemotherapeutic agents. Altogether, our study shows that inhibition of MCT1 or MCT4 impairs leukemic cell proliferation, suggesting that targeting lactate metabolism may be a new therapeutic strategy for AML, and points to MCT4 as a potential therapeutic target in AML patients and to syrosingopine as a new anti-proliferative drug and inducer of autophagy to be used in combination with conventional chemotherapeutic agents in AML treatment.

Early and Very Early GRIM19 and MCL1 Expression Are Correlated to Late Acquired Prednisolone Effects in a T-Cell Acute Leukemia Cell Line.

Glucocorticoids (GCs) are still first-line drugs for the treatment of childhood acute lymphoblastic leukemia (ALL). Prednisolone is a corticosteroid and one of the most important agents in the treatment of ALL. We report here a study of Prednisolone treatment using as a model a leukemia cell line with subsequent investigation of resistance-related gene expression. Gene silencing has been used in order to identify significant targets of resistance to GC-induced apoptosis in ALL cells. We analyzed effects of increasing doses of Prednisolone on ALL cell survival and growth, and we monitored immediate effects on gene expression through gene expression assays. We determined Prednisolone cytotoxicity and cell cycle distribution as well as DNA content. Upon treatment with escalating Prednisolone concentration, we observed a gradual decline in cell survival. MCL1 and GRIM19 were investigated as possible genes for the intrinsic capacity of this cell line to respond to corticosteroid and a snapshot of early changes was examined. Early MCL1 and GRIM19 expression correlated significantly to late GC-induced apoptosis. Prednisolone competitively induces MCL1 expression. Consistently with previous studies on primary leukemia blasts, cells are sensitive to proteasome inhibitor MG132; no interference of Prednisolone with MG132 effects on this cell line was noted. The inherent plasticity of clinically evolving cancer justifies approaches to characterize and prevent undesirable activation of early oncogenic pathways. Study of the pattern of intracellular signal pathway activation by anticancer drugs can lead to development of efficient treatment strategies by reducing detrimental secondary effects.