Primary Leukemia Cells Research Articles

Antiproliferative Activity of Cephalotaxus Esters: Overcoming Chemoresistance.

Omacetaxine, a semisynthetic form of Homoharringtonine (HHT), was approved for the treatment of Chronic Myeloid Leukemia (CML). Previously, we have published the synthesis of this natural alkaloid and three of its derivatives: Deoxyharringtonine (DHT), Deoxyhomoharringtonine (DHHT), and Bis(demethyl)-deoxyharringtonine (BDHT), and reported its refractory activity against the HL-60/RV+ cells over-expressing P-glycoprotein 1 (MDR1). In this study, we have explored the extent of this resistance by first expanding the panel of established cell lines and using a panel of 21 leukemia patient-derived primary cells. Herein, we have reported consistent resistance to HTT of K562-derived cells and to mitoxantrone of MES-SA/MX2-derived cells; all of them have been found to overexpress MDR1, while we have found U87MG-ABCG2 and H69AR cells to be very sensitive to HTT. In contrast, DHT, DHHT, and BDHT seemingly overcame this resistance due to the changes made to the acyl chain of HTT, rendering the derivatives less susceptible to efflux. Surprisingly, the leukemia primary cells were very sensitive to HHT and its derivatives with low nanomolar potencies, followed by a new class of CDC7 kinase inhibitors, the anthracycline class of topoisomerase inhibitors, the DNA intercalator actinomycin-D, and the vinca alkaloid class of microtubule inhibitors. The mechanism of cell death induced by HTT and DHHT was found to be mediated via caspase 3 cleavage, leading to apoptosis. Taken together, our results confirm that HHT is a substrate for MDR1. It opens the door to a new opportunity to clinically evaluate HHT and its derivatives for the treatment of AML and other cancers.

Pharmacological targeting of CBX7 alters the epigenetic landscape and induces differentiation of leukemic cells

AbstractSelf-renewal of leukemic cells results in the accumulation of dysfunctional blood cells and suppression of normal hematopoiesis. The polycomb group protein chromobox 7 (CBX7) is an epigenetic regulator that represses genes required for differentiation and cell cycle arrest and thereby promotes self-renewal. Because leukemic cells are highly self-renewing, we tested whether pharmacological targeting of CBX7 would reduce self-renewal and induce differentiation of human leukemic cells. We found that existing and newly developed CBX7 inhibitors derepress the epigenome, resulting in reduced ubiquitination of histone 2A and reduced binding of CBX7 to its target genes. This led to reduced cell growth, increased differentiation of leukemic cells in vitro, and delayed engraftment of primary leukemic cells in xenotransplant models. Therefore, pharmacological targeting of CBX7 constitutes a novel therapeutic approach for leukemia.

Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.

Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, ‘CLICK-on-CUT&Tag’, we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.

JR-AB2-011 induces fast metabolic changes independent of mTOR complex 2 inhibition in human leukemia cells.

The mechanistic target of rapamycin (mTOR) is a crucial regulator of cell metabolic activity. It forms part of several distinct protein complexes, particularly mTORC1 and mTORC2. The lack of specific inhibitors still hampers the attribution of mTOR functions to these complexes. JR-AB2-011 has been reported as a specific mTORC2 inhibitor preventing mTOR binding to RICTOR, a unique component of mTORC2. We aimed to describe the effects of JR-AB2-011 in leukemia/lymphoma cells, where the mTOR pathway is often aberrantly activated. The impact of JR-AB2-011 on leukemia/lymphoma cell metabolism was analyzed using the Seahorse platform. AKT phosphorylation at Ser473 was used as a marker of mTORC2 activity. mTOR binding to RICTOR was assessed by co-immunoprecipitation. RICTOR-null cells were derived from the Karpas-299 cell line using CRISPR/Cas9 gene editing. In leukemia/lymphoma cell lines, JR-AB2-011 induced a rapid drop in the cell respiration rate, which was variably compensated by an increased glycolytic rate. In contrast, an increase in the respiration rate due to JR-AB2-011 treatment was observed in primary leukemia cells. Unexpectedly, JR-AB2-011 did not affect AKT Ser473 phosphorylation. In addition, mTOR did not dissociate from RICTOR in cells treated with JR-AB2-011 under the experimental conditions used in this study. The effect of JR-AB2-011 on cell respiration was retained in RICTOR-null cells. JR-AB2-011 affects leukemia/lymphoma cell metabolism via a mechanism independent of mTORC2.

Overexpression of RBM4 promotes acute myeloid leukemia cell differentiation by regulating alternative splicing of TFEB

Alternative splicing (AS) is an efficient and ubiquitous transcriptional regulatory mechanism that expands the coding capacity of the genome and is associated with the occurrence and progression of cancer. The differentiation-promoting regimen is a potential therapeutic approach in cancer treatment. In this study, we screened NPMc-positive and NPMc-negative AML samples from the Cancer Genome Atlas (TCGA), focusing on the splicing factor RBM4 and its splicing mechanism on the target gene TFEB, which are most relevant to the prognosis of AML. We also investigated the impact of the TFEB-dominant spliceosome on autophagy and differentiation of THP-1 and K562 cells. The results showed that RBM4 recognized the CU-rich sequence in intron 8 of TFEB, increasing the production of the TFEB-L spliceosome, which promoted autophagy. Overexpression of RBM4 increased autophagy and promoted cell differentiation. The combination of TFEB-L with the therapeutic drug rapamycin further promoted the differentiation of leukemia cell lines and primary leukemia cells in AML patients. This study suggested that overexpression of RBM4 could promote cell differentiation by promoting the production of the TFEB-dominant spliceosome, demonstrating the potential of the TFEB-dominant spliceosome combined with chemotherapy drugs to promote leukemia cell differentiation and improve patient prognosis.

NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody

NK cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9-ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared to anti-NKG2A antibody blockade, NKG2A-knockout NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2A-deficient NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody-coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.

Lysosome-Disrupting Agents in Combination with Venetoclax Increase Apoptotic Response in Primary Chronic Lymphocytic Leukemia (CLL) Cells Mediated by Lysosomal Cathepsin D Release and Inhibition of Autophagy.

Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.

Novel meriolin derivatives potently inhibit cell cycle progression and transcription in leukemia and lymphoma cells via inhibition of cyclin-dependent kinases (CDKs)

A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs). Consequently, targeting CDKs via meriolins represents an attractive therapeutic approach for cancer therapy. Meriolins represent a semisynthetic compound class derived from meridianins and variolins with a known CDK inhibitory potential. Here, we analyzed the two novel derivatives meriolin 16 and meriolin 36 in comparison to other potent CDK inhibitors and could show that they displayed a high cytotoxic potential in different lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells. In a kinome screen, we showed that meriolin 16 and 36 prevalently inhibited most of the CDKs (such as CDK1, 2, 3, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20). In drug-to-target modeling studies, we predicted a common binding mode of meriolin 16 and 36 to the ATP-pocket of CDK2 and an additional flipped binding for meriolin 36. We could show that cell cycle progression and proliferation were blocked by abolishing phosphorylation of retinoblastoma protein (a major target of CDK2) at Ser612 and Thr82. Moreover, meriolin 16 prevented the CDK9-mediated phosphorylation of RNA polymerase II at Ser2 which is crucial for transcription initiation. This renders both meriolin derivatives as valuable anticancer drugs as they target three different Achilles’ heels of the tumor: (1) inhibition of cell cycle progression and proliferation, (2) prevention of transcription, and (3) induction of cell death.

Minnelide exhibits antileukemic activity by targeting the Ars2/miR-190a-3p axis

BackgroundThe identification of a novel and effective strategy for the clinical treatment of acute leukemia (AL) is a long-term goal. Minnelide, a water-soluble prodrug of triptolide, has recently been evaluated in phase I and II clinical trials in patients with multiple cancers and has shown promise as an antileukemic agent. However, the molecular mechanism underlying minnelide's antileukemic activity remains unclear. PurposeTo explore the molecular mechanisms by which minnelide exhibits antileukemic activity. MethodsAL cells, primary human leukemia cells, and a xenograft mouse model were treated with triptolide and minnelide. The molecular mechanism was elucidated using western blotting, immunoprecipitation, flow cytometry, GSEA and liquid chromatography-mass spectrometry analysis. ResultsMinnelide was highly effective in inhibiting leukemogenesis and improving survival in two complementary AL mouse models. Triptolide, an active form of minnelide, causes cell cycle arrest in G1 phase and induces apoptosis in both human AL cell lines and primary AL cells. Mechanistically, we identified Ars2 as a new chemotherapeutic target of minnelide for AL treatment. We found that triptolide directly targeted Ars2, resulting in the downregulation of miR-190a-3p, which led to the disturbance of PTEN/Akt signaling and culminated in G1 cell cycle arrest and apoptosis. ConclusionsOur findings demonstrate that targeting Ars2/miR-190a-3p signaling using minnelide could represent a novel chemotherapeutic strategy for AL treatment and support the evaluation of minnelide for the treatment of AL in clinical trials.

A chemically adjustable BMP6-IL6 axis in mesenchymal stem cells drives acute myeloid leukemia cell differentiation

Chemotherapy alone or in combination with allogeneic stem cell transplantation has been the standard of care for acute myeloid leukemia (AML) for decades. Leukemia relapse with limited treatment options remains the main cause of treatment failure. Therefore, an effective and safe approach to improve treatment outcomes is urgently needed for most AML patients. Mesenchymal stem cells (MSCs) have been reported to efficiently induce apoptosis and shape the fate of acute myeloid leukemia cells. Here, we identified LG190155 as a potent compound that enhances the antileukemia efficiency of MSCs. Pretreatment of MSCs with LG190155 significantly provoked differentiation in both AML patient-derived primary leukemia cells and AML cell lines and reduced the tumor burden in the AML mouse model. Using the quantitative proteomic technique, we discovered a pivotal mechanism that mediates AML cell differentiation, in which autocrine bone morphogenetic protein 6 (BMP6) in MSCs boosted IL-6 secretion and further acted on leukemic cells to trigger differentiation. Furthermore, the activity of the BMP6-IL6 axis was dramatically enhanced by activating vitamin D receptor (VDR) in MSCs. Our data illustrated an effective preactivated approach to reinforcing the antileukemia effect of MSCs, which could serve as an effective therapeutic strategy for AML.

Prognostic Value of IGF2BP3 Gene Expression Levels in Patients with Acute Myeloid Leukemia

To investigate the relationship between IGF2BP3 gene expression and prognosis in patients with acute myeloid leukemia (AML). High throughput transcriptome sequencing was performed on bone marrow primary leukemia cells from 27 patients with AML in our center, the relationship between IGF2BP3 expression levels and clinical characteristics were analyzed and verify the samples from patients with newly treated AML and refractory AML. The expression level of IGF2BP3 gene were analyzed in 20 healthy subjects and 26 patients with AML. The expression of IGF2BP3 in two anthracycline-resistant cell lines (HL60/ADR, K562/ADR) was detected by RT-qPCR and Western blot, and the expression difference of IGF2BP3 was compared with that in sensitive cells (HL60, K562). The relationship between the expression level of IGF2BP3 in patients with AML and prognostic were analyzed through data analysis of 746 patients with AML, and the prognostic value of IGF2BP3 in AML was analyzed by multivariate Cox regression analysis. In the bone marrow primary leukemia cells of 27 AML patients in our center, the expression level of IGF2BP3 in patients with refractory AML was significantly higher than that in chemotherapy sensitive patients (P =0.0343). The expression of IGF2BP3 in leukemia patients with extramedullary infiltration (EMI) was significantly higher than that in AML patients without extramedullary infiltration (P =0.0049). Compared with healthy subjects (n=20), IGF2BP3 expression in AML patients (n=26) was higher (P =0.0009). The expression of IGF2BP3 mRNA in the anthracycline resistant cell lines (HL60/ADR, K562/ADR) was significantly higher than that in the sensitive cell lines (K562/ADR vs K562,P =0.0430; HL60/ADR vs HL60, P =0.7369). Western blot results showed that the expression of IGF2BP3 protein in mycin resistant cells was significantly higher than that in sensitive cells (P < 0.001). qPCR results showed that the expression level of IGF2BP3 mRNA in refractory AML patients was significantly higher than that in patients with chemotherapy sensitive (P =0.002). High expression of IGF2BP3 was associated with poor prognosis in AML (P < 0.05) in 3 large sample cohorts of AML patients. Univariate and multivariate prognostic analyses demonstrated that high expression of IGF2BP3 was significantly associated with shorter event-free survival (EFS, HR=1.887, P =0.024) and overall survival (OS, HR=1.619, P =0.016). The high expression of IGF2BP3 gene may be an important factor in the poor prognosis of AML, suggesting that IGF2BP3 gene may be a new molecular marker for the clinical prognosis evaluation and treatment strategy of AML.

Abstract 187: Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia

Abstract BCR-ABL1 fusion gene is the hall marker of chronic myeloid leukemia (CML). Currently, the immune microenvironment of BCRABL1 CML remains largely unexplored. Our recent investigation in a transgenic mouse model of BCRABL1 CML revealed a continuous increase in CD11b+Ly6Cint PMN-MDSC with the progression of the disease. Concurrently, there was an increase in CD11b+Ly6Chigh M-MDSC and F4/80+ macrophages. However, The levels of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD49b+ NK cells, which are essential for tumor-killing significantly decreased. This immune phenotype was consistently observed in both primary leukemia induction and following serial transplantation of cells from primary leukemic mice. Intriguingly, only recipient mice subjected to sublethal irradiation developed the disease, while mice with intact immune system remained disease-free, which underscores the vital role of the immune system in the development of CML. The predominant presence of CD11b+Ly6Cint PMN-MDSC in the peripheral blood, bone marrow and spleen implies these cells may be at least partially consisted of CML leukemia cells. To examine the hypothesis, qRT-PCR and immunofluorescence (IF) staining were conducted. In both assays, Ly6G+ cells from BCRABL1 mice coexpressed the BCRABL1 oncogene. Furthermore, when co-cultured with CD4 T cells, the division of T cells was significantly inhibited, implying immune suppression by the Ly6G+ PMN-MDSCs and/or CML leukemia cells. In addition, the immuosuppressive pathway genes including Arg1, Hif1a, Enptd1, TGFβ were significantly upregulated. To investigate the possible role of the PD1/PDL1 axis in the BCRABL1 model, flow cytometry analysis was conducted. Unexpectedly, PDL1 was barely expressed in CML leukemia cells, whereas the expression of PDL1 in M-MDSCs and macrophages significantly increased, accompanied by remarkable upregulation of PD1 expression on CD4 T cells and CD8 T cells during leukemia progression. In other words, PDL1 expression on macrophages, rather than leukemia cells, was found to induce T cell exhaustion. For translational purpose, mice transplanted with the primary CML leukemia cells were treated with chemotherapy drugs ponatinib alone or in combination with the anti-PD1 antibody. Compared with the chemotherapy alone group, the mice in the combinational therapy group exhibited rapid remission and better disease control. Most importantly, the disease relapse occurred at a slower rate after treatment discontinuation, suggesting the value of combinational therapy in achieving treatment free remission. In summary, our study not only revealed the dynamic changes of immune microenvironment in the BCRABL1 CML mouse model, but also revealed the different mechanisms underlying immune evasion of BCR-ABL1 CML leukemia cells, which sheds light on a combinational treatment for achieving the treatment free remission of CML. Citation Format: Xiaocui Lu, Hui Fang, Xuexiu Fang, Atsuko Matsunaga, Kebin Liu, Jorge Cortes, John K. Cowell, Tianxiang Hu. Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 187.

Abstract 5956: FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients

Abstract Backgroud: The prognosis of B cell ALL patients (pts) relapsing after Hematopoietic Transplant is poor and new drugs are needed. In acute myeloid leukemia, FLT3-inhibitors (FLT3i) are showing promising results in FLT3-mutated (mut) cases. Their potential use in FLT3-mut B-ALL has been poorly investigated. Aim: To assess if FLT3 could be a valid targetable marker in B-ALL. Methods: We sequenced with a capture based large RNA-seq panel (1385genes) 183 adult B-ALL [n=52 Pos for Ph or t(4;11) or t(1;19) and n=131 Triple-Negative (TN) pts that are negative for Ph (n=43), t(4;11) and t(1;19) translocations] and 15 donors. To confirm FLT3 muts, 13 available samples were further sequenced with Extended Myeloid Solution (98 genes; SOPHiA) where FLT3 gene is fully covered. In vitro studies using 5 FLT3i [Gilteritinib (Gil), Midostaurin, Crenolanib, Sorafenib and Quizartibin] and Venetoclax (Ven) were conducted on pt primary cells and on TN and other B-ALL cell lines (MUTZ5, MHH-CALL4 Ph-like; NALM6;RS4;11, 697, REH) and 2 AML cell lines (OCI-AML3 FLT3;MV-4-11 FLT3-ITD). Results: We found 15 FLT3 mut in 14/131 TN B-ALL and in 1/43 Ph+ cases: 43.8% were TKD mut, 3 ITD mut, 3 splicing site mut,1 N-terminal, 1 juxta-membrane domain and 1 Ig-like site (Fig. 1A). After pathogenicity interpretation we excluded 3 variants and one mutation was not confirmed at DNA level. Overall 7.6% of TN were mut (Ph+ 2.3%). 81.8% of FLT3mut pts potentially druggable with FLT3i, mainly TKD (63.3%). Targeted NGS revealed the co-occurrence with other mutations (range 2-15), and in particular with CSMD1 (n=4), KDM6A, KMT2D and CREBBP (n=3) alterations. Moreover, 11/13 samples carried copy number alteration (mean 12), mostly amplifications. CDKN2A &amp; IKZF1 were the most frequently altered genes (n=5;4 CN-loss). FLT3 expression was increased in 11/15 cases compared to donors and wt pts. As reported, the expression was higher in ZNF384 or MLLr B-ALL subtypes.To evaluate the effect of FLT3is in ALL, we treated ex vivo primary leukemic cells of 6 adult ALL patients (FLT3-mut n=4; FLT3-wt n=2) with increasing concentrations of 5 FLT3i for 24, 48 and 72h. We observed a trend towards greater response of FLT3-mut ALL cells compared to wt ones. The average IC50 values in response to the five FLT3i was 1.6 uM and 24.5 uM after 72h of treatment in FLT3-mut and FLT3-wt B-ALL, respectively (Fig. 1B). We than evaluated the sensitivity to the 5 Inhs in all cell lines. Interestingly MHH-CALL4 wt were more sensitive to Gil compared to OCI AML3 wt, and showed the same effect compare to MV-4-11 AML mut (F.1C). Given the effect of Gil in single agent, we combined it with Ven in wt B-ALL cells at increasing concentrations. Interestingly We noticed a strong additive effect with the higher Gil concentration and all Ven doses(F.1D). Conclusions: FLT3 alterations identify a novel subgroup of TN B-ALL with therapeutic potential also in combination regimens. Ricerca Corrente Italian MoH L3P1946. Citation Format: Anna Ferrari, Andrea Ghelli Luserna di Rorà, Federico Lo Schiavo, Eugenio Fonzi, Giorgia Capirossi, Chiara Salvesi, Maria Teresa Bochicchio, Lorenzo Ledda, Chiara Servilli, Matteo Paganelli, Giovanni Marconi, Cristina Papayannidis, Chiara Sartor, Michela Rondoni, Anna Maria Mianulli, Barbara Giannini, Crescenza Pasciolla, Fabio Giglio, Sara Galimberti, Monica Fumagalli, Prassede Salutari, Valentina Gaidano, Giovanni Martinelli, Giorgia Simonetti. FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5956.

Pharmacological degradation of ATR induces antiproliferative DNA replication stress in leukemic cells.

Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.

Abstract 4710: The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism

Abstract Background: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are devastating blood cancers associated with 5-year survival rates of ~50% each in adults. We recently developed a new class of polymeric drugs called PAMDs based on the CXCR4 inhibitor AMD3100 (plerixafor). PAMDs have the potential to be more effective and less toxic therapeutics than the standard of care high-dose chemotherapy. We tested one variant (PAMD-Ch17), and found that unlike AMD3100, PAMD-Ch17 has several novel anti-leukemic effects. To understand the potential mechanism(s) for these new activities, we performed the following study. Methods: To test the role of CXCR4 in PAMD-Ch17’s activities, we generated CXCR4 knockout cells via Crispr/Cas9. We used PrestoBlue, 7-AAD, and Annexin V staining to assay viability and apoptosis. To identify pathways deregulated by PAMD-Ch17, we performed whole transcriptomic sequencing (RNA-Seq). To investigate if PAMD-Ch17 mediates its activities by inducing oxidative stress, we performed SeaHorse assays, and dihydroethidium (DHE), Mitosox, and Bodipy C11 staining. To investigate whether PAMD induces programmed cell death pathways, we used ferrostatin and deferoxamine to inhibit ferroptosis, necrostatin-1 against necroptosis, and Z-VAD-FMK against apoptosis. Results: We found that PAMD-Ch17 induces cell death in a dose dependent manner in human AML and ALL cell lines and, mouse primary leukemia cells, but not healthy mouse bone marrow cells. Surprisingly, PAMD-Ch17 induces equivalent levels of cell death in both wild type and CXCR4 knockout Jurkat cells, indicating that the only known target of the polymer is not required for its anti-leukemic effects. By RNA-Seq, we found altered expression of genes related to mitochondrial function, suggesting that PAMD-Ch17 could be targeting the mitochondria. To test this, we performed SeaHorse assays and found that PAMD-Ch17 induces a significant decrease in mitochondrial respiration. Consistent with this, we found that PAMD-Ch17 induces a significant increase in superoxide production, as well as lipid peroxidation. Using inhibitors of programmed cell death pathways, we found that preventing necroptosis, apoptosis, or ferroptosis caused only subtle effects on PAMD-Ch17 induced cell death, implying that the polymer’s anti-leukemic activity is likely not entirely dependent on these pathways. Conclusions: These results indicate that PAMD-Ch17 induces cell death in leukemia cells but not healthy bone marrow via a CXCR4 independent mechanism. We also found that PAMD-Ch17 induces reactive oxygen species, potentially explaining its selectivity for leukemia cells compared to healthy blood cells. Continued investigation of PAMD-Ch17 will not only further development of this new class of drugs, but also provide much needed insight into leukemia biology. Citation Format: Calvin Lam, Jogdeo Chinmay, Ekta Kapoor, Siyuan Tang, Svetlana Romanova, David Oupicky, Katherine Hyde. The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4710.

Disrupting pro-survival and inflammatory pathways with dimethyl fumarate sensitizes chronic lymphocytic leukemia to cell death

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells.

Analysis of Primary Chronic Lymphocytic Leukemia Cells' Signaling Pathways.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

ETV6::ACSL6 translocation-driven super-enhancer activation leads to eosinophilia in acute lymphoblastic leukemia through IL-3 overexpression.

ETV6::ACSL6 represents a rare genetic aberration in hematopoietic neoplasms and is often associated with severe eosinophilia, which confers an unfavorable prognosis requiring additional anti-inflammatory treatment. However, since the translocation is unlikely to produce a fusion protein, the mechanism of ETV6::ACSL6 action remains unclear. Here, we performed multi-omics analyses of primary leukemia cells and patient-derived xenografts from an acute lymphoblastic leukemia (ALL) patient with ETV6::ACSL6 translocation. We identified a super-enhancer located within the ETV6 gene locus, and revealed translocation and activation of the super-enhancer associated with the ETV6::ACSL6 fusion. The translocated super-enhancer exhibited intense interactions with genomic regions adjacent to and distal from the breakpoint at chromosomes 5 and 12, including genes coding inflammatory factors such as IL-3. This led to modulations in DNA methylation, histone modifications, and chromatin structures, triggering transcription of inflammatory factors leading to eosinophilia. Furthermore, the bromodomain and extraterminal domain (BET) inhibitor synergized with standard-of-care drugs for ALL, effectively reducing IL-3 expression and inhibiting ETV6::ACSL6 ALL growth in vitro and in vivo. Overall, our study revealed for the first time a cis-regulatory mechanism of super-enhancer translocation in ETV6::ACSL6ALL, leading to an ALL-accompanying clinical syndrome. These findings may stimulate novel treatment approaches for this challenging ALL subtype.

Generation of a new therapeutic d-peptide that induces the differentiation of acute myeloid leukemia cells through A TLR-2 signaling pathway

Acute myeloid leukemia (AML) is caused by clonal disorders of hematopoietic stem cells. Differentiation therapy is emerging as an important treatment modality for leukemia, given its less toxicity and wider applicable population, but the arsenal of differentiation-inducing agents is still very limited. In this study, we adapted a competitive peptide phage display platform to search for candidate peptides that could functionally induce human leukemia cell differentiation. A monoclonal phage (P6) and the corresponding peptide (pep-P6) were identified. Both l- and d-chirality of pep-P6 showed potent efficiency in inducing AML cell line differentiation, driving their morphologic maturation and upregulating the expression of macrophage markers and cytokines, including CD11b, CD14, IL-6, IL-1β, and TNF-α. In the THP-1 xenograft animal model, administration of d-pep-P6 was effective in inhibiting disease progression. Importantly, exposure to d-pep-P6 induced the differentiation of primary human leukemia cells isolated AML patients in a similar manner to the AML cell lines. Further mechanism study suggested that d-pep-P6 induced human leukemia cell differentiation by directly activating a TLR-2 signaling pathway. These findings identify a novel d-peptide that may promote leukemia differentiation therapy.