Mediated Cytotoxicity Research Articles

Abstract 723: Reduction of immunosuppressive function of regulatory T cells enhances antigen-specific T cell mediated cytotoxicity against tumor cells

Abstract Regulatory T cells (Tregs) maintain immune homeostasis by suppressing abnormal immune response to self and non-self-antigens. Tregs are also involved in tumor development and progression by inhibiting anti-tumor immunity. Infiltration of Tregs in the tumor microenvironment (TME) inhibit anti-tumor immunity by suppressing tumor antigen-specific T cell responses. This results in tumor progression and poor prognosis in patients with various types of cancers. Therefore, depletion of Tregs could be a promising immunotherapy target to augment the anti-tumor immune response. Molecules highly expressed by Tregs such as CD25, immune checkpoint molecules, and chemokine receptors have been targeted by antibodies or small molecules to deplete Tregs and its immunosuppressive function has been tested in the clinic. However, a thin line exists between augmenting effective immunity and inducing autoimmunity, posing difficulties in harnessing Tregs modulation in cancer treatment. Thus, additional strategies to selectively control Tregs are urgently needed for the effective treatment of cancer. In this study we evaluated the role of Tregs in suppressing the effector function of antigen-specific T cells and targeted killing of cancer cells in vitro. We observed Tregs mediated suppression of antigen-specific cytotoxicity against cancer cells. When Tregs were treated with cyclophosphamide, CD25 depleting antibody and anti-CTLA4 antibody alone or in combination, we observed enhanced killing of cancer cells in vitro as a result of potentiated effector function of antigen-specific T cells. This study provides a path forward to fine-tune and optimize strategies to target various molecules that are highly expressed on Tregs and augment anti-tumor immunity by antigen-specific T cells to achieve therapeutic outcome. Citation Format: Arif Azam Khan, Jaehyung Park, Nate LaVoy, Chris Tompkins, Jeff Bellinder, Tisha San Miguel, Neal Lawrence, Brianna Schoen. Reduction of immunosuppressive function of regulatory T cells enhances antigen-specific T cell mediated cytotoxicity against tumor cells [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 723.

Abstract 2736: Ligand-independent targeting of tumor cells for antibody-dependent cell-mediated cytotoxicity

Abstract Introduction: Biological targeting is a key concept in cancer treatment utilized by most therapeutics. Targeted agents that selectively treat diseased tissues while reducing off-target effects to normal tissues are widely used for cancer therapies. However, key hurdles include: (i) a limited number of tumor-specific biomarkers expressed in sufficiently high levels for targeted therapy, (ii) lack of specific biomarkers amenable to targeting, and (iii) current technologies rely on largely protein-based targeting ligands limiting the applications of these therapies due to final conjugates with low payloads. We developed a novel method for tumor-specific delivery by combining metabolic glycoengineering and nanomedicine to engineer targetable chemical groups on cancer cells and enable directed treatments. We hypothesize that increased Fc fragments on the surface of tumor cells can enable antibody-dependent cell mediated cytotoxicity (ADCC) response to tumor cells, providing innate immunotherapy. Materials/Methods: We formulated nanoparticles (NPs) using polyethylene glycol-ply lactic acid-co-glycolic acid (PEG-PLGA). Using the Dolomite microfluidic device, we formed NPs by nanoprecipitation of PEG-PLGA with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), a sialic acid analogue containing a click chemistry group. We targeted this reactive group by functionalizing the Fc fragment with dibenzylcyclooctyne (DBCO). We characterized the NPs using dynamic light scattering (DLS), charge measurements and transmission electron microscopy (TEM). We evaluated toxicity using a cell viability assay and assessed preliminary efficacy with a tumor growth delay study. Results: NP characterization revealed a size of 30-40 nm via DLS, congruent with TEM imaging. Charge measurements ranged from -4 to +4 mV. Toxicity studies revealed no differences in cell viability amongst different treatment groups. Preliminary efficacy studies demonstrated improved tumor growth delay in the cohort of mice treated with a combination of NPs and Fc-DBCO conjugate, as well as a survival benefit. Median OS was 31.5 days for the control groups vs 57.5 days for the combination treated group (p = 0.0067). Conclusion: There is growing interest in harnessing the power of the innate immune response to shape anti-tumor immunity. This is because clinical data demonstrates tumor cells can evade the adaptive immune system through mutations as well as downregulate MHC receptors. One of the most effective innate immune responses is ADCC. To this end, we developed a novel method for tumor-specific delivery of Fc fragments to enhance ADCC. While this data reveals improved tumor growth delay and a survival benefit, the sample size is limited, and a larger study is needed to evaluate the potential benefits of this strategy. Furthermore, correlative studies are needed to identify the mechanism for treatment response. Citation Format: Maureen L. Aliru, Nikhila R. Sultanpuram, Kin Man Au, Tian Zhang, Andrew Z. Wang. Ligand-independent targeting of tumor cells for antibody-dependent cell-mediated cytotoxicity [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 2736.

Identification and characterization of circular RNAs expression profiles in obstructive sleep apnea-induced liver injury.

Circular RNAs (circRNAs) have exhibited microRNA sponge activity, related to many important biological processes. Our study attempted to explore the comprehensive changes of circRNAs expression pattern in Obstructive sleep apnea (OSA)-induced liver injury and provide a global perspective of differentially expressed circRNAs (DECs). Then, RT-qPCR was used to confirm the microarray data. Further, gene ontology (GO) and KEGG pathway analysis were performed to annotate the DECs. Finally, the circRNA-miRNA-mRNA interaction network was established to predicted the target genes and target miRNAs of DECs for a stepwise bioinformatics analysis. We revealed a total of eighty DECs. In the meantime, six circRNAs were randomly validated by RT-qPCR. Among these circRNAs, mmu_circRNA_000469, 37851, 38959, 38983, 31665 were up-regulated in both microarray and qRT-PCR tissues, while mmu_circRNA_27565 was down-regulated. GO analysis revealed that circRNAs-target genes were largely related to liver function process such as carboxylic acid metabolic process and negative regulation of mitochondrial membrane potential. Meanwhile, KEGG analysis found that there were 13 pathways related to these circRNAs- target genes. And the most enriched pathway was Natural killer cell mediated cytotoxicity, which strongly suggests that immune responses may be important for the process of OSA-induced liver injury. In addition, four significant DECs (mmu_circRNA_000469, 38959, 38983, 27565) and their target mRNA and target miRNAs were further selected to establish the regulation network. Our study revealed that circRNAs may play a crucial role in OSA-induced liver injury and thus mmu_circRNA_000469, 38959, 38983, 27565 may serve as biomarkers of biological process of OSA-induced liver injury.

Multi-omics integration identifies regulatory factors underlying bovine subclinical mastitis

BackgroundMastitis caused by multiple factors remains one of the most common and costly disease of the dairy industry. Multi-omics approaches enable the comprehensive investigation of the complex interactions between multiple layers of information to provide a more holistic view of disease pathogenesis. Therefore, this study investigated the genomic and epigenomic signatures and the possible regulatory mechanisms underlying subclinical mastitis by integrating RNA sequencing data (mRNA and lncRNA), small RNA sequencing data (miRNA) and DNA methylation sequencing data of milk somatic cells from 10 healthy cows and 20 cows with naturally occurring subclinical mastitis caused by Staphylococcus aureus or Staphylococcus chromogenes.ResultsFunctional investigation of the data sets through gene set analysis uncovered 3458 biological process GO terms and 170 KEGG pathways with altered activities during subclinical mastitis, provided further insights into subclinical mastitis and revealed the involvement of multi-omics signatures in the altered immune responses and impaired mammary gland productivity during subclinical mastitis. The abundant genomic and epigenomic signatures with significant alterations related to subclinical mastitis were observed, including 30,846, 2552, 1276 and 57 differential methylation haplotype blocks (dMHBs), differentially expressed genes (DEGs), lncRNAs (DELs) and miRNAs (DEMs), respectively. Next, 5 factors presenting the principal variation of differential multi-omics signatures were identified. The important roles of Factor 1 (DEG, DEM and DEL) and Factor 2 (dMHB and DEM), in the regulation of immune defense and impaired mammary gland functions during subclinical mastitis were revealed. Each of the omics within Factors 1 and 2 explained about 20% of the source of variation in subclinical mastitis. Also, networks of important functional gene sets with the involvement of multi-omics signatures were demonstrated, which contributed to a comprehensive view of the possible regulatory mechanisms underlying subclinical mastitis. Furthermore, multi-omics integration enabled the association of the epigenomic regulatory factors (dMHBs, DELs and DEMs) of altered genes in important pathways, such as ‘Staphylococcus aureus infection pathway’ and ‘natural killer cell mediated cytotoxicity pathway’, etc., which provides further insights into mastitis regulatory mechanisms. Moreover, few multi-omics signatures (14 dMHBs, 25 DEGs, 18 DELs and 5 DEMs) were identified as candidate discriminant signatures with capacity of distinguishing subclinical mastitis cows from healthy cows.ConclusionThe integration of genomic and epigenomic data by multi-omics approaches in this study provided a better understanding of the molecular mechanisms underlying subclinical mastitis and identified multi-omics candidate discriminant signatures for subclinical mastitis, which may ultimately lead to the development of more effective mastitis control and management strategies.

Antioxidant Enzymes in Cancer Cells: Their Role in Photodynamic Therapy Resistance and Potential as Targets for Improved Treatment Outcomes.

Photodynamic therapy (PDT) is a selective tumor treatment that consists of a photosensitive compound-a photosensitizer (PS), oxygen, and visible light. Although each component has no cytotoxic properties, their simultaneous use initiates photodynamic reactions (PDRs) and sequentially generates reactive oxygen species (ROS) and/or free radicals as cytotoxic mediators, leading to PDT-induced cell death. Nevertheless, tumor cells develop various cytoprotective mechanisms against PDT, particularly the adaptive mechanism of antioxidant status. This review integrates an in-depth analysis of the cytoprotective mechanism of detoxifying ROS enzymes that interfere with PDT-induced cell death, including superoxide dismutase (SOD), catalase, glutathione redox cycle, and heme oxygenase-1 (HO-1). Furthermore, this review includes the use of antioxidant enzymes inhibitors as a strategy in order to diminish the antioxidant activities of tumor cells and to improve the effectiveness of PDT. Conclusively, PDT is an effective tumor treatment of which its effectiveness can be improved when combined with a specific antioxidant inhibitor.

Transcriptome profile analysis in spinal cord injury rats with transplantation of menstrual blood-derived stem cells.

Menstrual blood-derived stem cells (MenSCs) are vital in treating many degenerative and traumatic disorders. However, the underlying molecular mechanisms remain obscure in MenSCs-treating spinal cord injury (SCI) rats. MenSCs were adopted into the injured sites of rat spinal cords at day 7 post surgery and the tissues were harvested for total RNA sequencing analysis at day 21 after surgery to investigate the expression patterns of RNAs. The differentially expressed genes (DEGs) were analyzed with volcano and heatmap plot. DEGs were sequentially analyzed by weighted gene co-expression network, functional enrichment, and competitive endogenous RNAs (ceRNA) network analysis. Next, expression of selected miRNAs, lncRNAs, circRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics packages and extra databases were enrolled to scoop the genes functions and their interaction relationships. A total of 89 lncRNAs, 65 circRNAs, 120 miRNAs and 422 mRNAs were significantly upregulated and 65 lncRNAs, 72 circRNAs, 74 miRNAs, and 190 mRNAs were significantly downregulated in the MenSCs treated rats compared to SCI ones. Current investigation revealed that MenSCs treatment improve the recovery of the injured rats and the most significantly involved pathways in SCI regeneration were cell adhesion molecules, nature killer cell mediated cytotoxicity, primary immunodeficiency, chemokine signaling pathway, T cell receptor signaling pathway and B cell receptor signaling pathway. Moreover, the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA network of SCI was constructed. Finally, the protein-protein interaction (PPI) network was constructed using the top 100 DE mRNAs. The constructed PPI network included 47 nodes and 70 edges. In summary, the above results revealed the expression profile and potential functions of differentially expressed (DE) RNAs in the injured spinal cords of rats in the MenSCs-treated and SCI groups, and this study may provide new clues to understand the mechanisms of MenSCs in treating SCI.

Pro-fluorescent probe with morpholine moiety and its reactivity towards selected biological oxidants.

Biological oxidants participate in many processes in the human body. Their excessive production causes organelle damage, which may result in the accumulation of cytotoxic mediators and cell degradation and may manifest itself in various diseases. Peroxynitrite (ONOO- ), hypochlorous acid (HOCl), hydrogen peroxide (H2 O2 ), and peroxymonocarbonate (HOOCO2 - ) are important oxidants in biology, toxicology, and various pathologies. Derivatives of coumarin, containing an oxidant-sensitive boronate group, have been recently developed for the fluorescent detection of inflammatory oxidants. Here, we report the synthesis and characterization of 4-[2-(morpholin-4-yl)-2-oxoethyl]-2-oxo-2H-chromen-7-yl boronic acid (MpC-BA) as a fluorescent probe for the detection of oxidants, with better solubility in water, high stability and fast response time toward peroxynitrite and hypochlorous acid. The effectiveness of the MpC-BA probe for the detection of peroxynitrite was measured by adding bolus ONOO- or using the co-generating superoxide and nitrogen oxide system. MpC-BA is oxidized by ONOO- to 7-hydroxy-4-[2-(morpholin-4-yl)-2-oxoethyl]-2H-chromen-2-one (MpC-OH). However, peroxynitrite-specific product (MpC-H) is formed in the minor reaction pathway. MpC-OH is also yielded in the reaction of MpC-BA with HOCl, and the subsequent formation of a chlorinated MpC-OH gives a specific product for HOCl (MpC-OHCl). H2 O2 slowly oxidizes MpC-BA. However, the addition of NaHCO3 increased the MpC-OH formation rate. We conclude that MpC-BA is potentially an improved fluorescent probe detecting peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of chlorinated and reduced coumarin(s) will help identify the oxidants detected.

Identification of key genes in chronic intermittent hypoxia-induced lung cancer progression based on transcriptome sequencing

BackgroundObstructive sleep apnea (OSA) is associated with increased risk of lung cancer mortality. Nevertheless, little is known about the underlying molecular mechanisms. This research aimed to investigate differentially expressed genes (DEGs) and explore their function in Lewis lung carcinoma (LLC)-bearing mice exposed to chronic intermittent hypoxia (CIH) by transcriptome sequencing.MethodsLung cancer tissues in LLC-bearing mice exposed to CIH or normoxia were subjected for transcriptome sequencing to examine DEGs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were employed to explore the function of DEGs. To evaluate the prognostic value of DEGs, the Kaplan–Meier survival analysis in combination with Cox proportional hazard model were applied based on The Cancer Genome Atlas.ResultsA total of 388 genes with 207 up-regulated and 181 down-regulated genes were differentially expressed between the CIH and normoxia control groups. Bioinformatics analysis revealed that the DEGs were related to various signaling pathways such as chemokine signaling pathway, IL-17 signaling pathway, TGF-β signaling pathway, transcriptional misregulation in cancer, natural killer cell mediated cytotoxicity, PPAR signaling pathway. In addition, the DEGs including APOL1, ETFB, KLK8, PPP1R3G, PRL, SPTA1, PLA2G3, PCP4L1, NINJ2, MIR186, and KLRG1 were proven to be significantly correlated with poorer overall survival in lung adenocarcinoma.ConclusionsCIH caused a significant change of gene expression profiling in LLC-bearing mice. The DEGs were found to be involved in various physiological and pathological processes and correlated with poorer prognosis in lung cancer.

Computational analysis of peripheral blood RNA sequencing data unravels disrupted immune patterns in Alzheimer's disease.

The central nervous system (CNS) and the immune system collectively coordinate cellular functionalities, sharing common developmental mechanisms. Immunity-related molecules exert an influence on brain development, challenging the conventional view of the brain as immune-privileged. Chronic inflammation emerges as a key player in the pathophysiology of Alzheimer's disease (AD), with increased stress contributing to the disease progression and potentially exacerbating existing symptoms. In this study, the most significant gene signatures from selected RNA-sequencing (RNA-seq) data from AD patients and healthy individuals were obtained and a functional analysis and biological interpretation was conducted, including network and pathway enrichment analysis. Important evidence was reported, such as enrichment in immune system responses and antigen processes, as well as positive regulation of T-cell mediated cytotoxicity and endogenous and exogenous peptide antigen, thus indicating neuroinflammation and immune response participation in disease progression. These findings suggest a disturbance in the immune infiltration of the peripheral immune environment, providing new challenges to explore key biological processes from a molecular perspective that strongly participate in AD development.

A lipo-polymeric hybrid nanosystem with metal enhanced fluorescence for targeted imaging of metastatic breast cancer.

Cancer metastasis plays a major role in failure of therapeutic avenues against cancer. Owing to metastasis, nearly 70-80% of stage IV breast cancer patients lose their lives. Nanodrug delivery systems are playing a critical role in the therapy of metastatic cancer in the recent times. This paper reports the enhanced permeation and retention (EPR) based targeting of metastatic breast cancer using a novel nano lipo-polymeric system (PIR-Au NPs). The PIR-Au NPs demonstrated an increase in fluorescence by virtue of surface coating with gold, owing to the metal enhanced fluorescence phenomenon as reported in our earlier reports. Enhanced fluorescence of PIR-Au NPs was observed in murine mammary carcinoma cell line (4T1), as compared to free IR780 or IR780 loaded nanosystems (P-IR NPs), when incubated for same time at same concentrations, indicating its potential application for imaging and an enhanced bioavailability of IR780. Significant cell death was noted with photothermal mediated cytotoxicity in-vitro against breast cancer cells (MCF-7 and 4T1). An enhanced fluorescence was observed in the zebra fish embryos incubated with PIR-Au NPs. The enhanced permeation and retention (EPR) effect was seen with PIR-Au NPs in-vivo. A strong fluorescent signal was recorded in mice injected with PIR-Au NPs. The tumor tissue collected after 72 h, clearly showed a greater fluorescence as compared to other groups, indicating the plasmon enhanced fluorescence. We also demonstrated the EPR-based targeting of the PIR-Au NPs in-vivo by means of photothermal heat. This lipo-polymeric hybrid nanosystem could therefore be successfully applied for image-guided, passive-targeting to achieve maximum therapeutic benefits.

IGSF8 on Cancer Cells Suppresses NK Cell–Mediated Cytotoxicity

IGSF8 on Cancer Cells Suppresses NK Cell–Mediated Cytotoxicity

Transcriptomic responses to RGNNV and Vibrio alginolyticus infection in the spleen of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀)

The hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) is a novel and commercially important fish for mariculture in China. Recent years, the outbreak of viral and bacterial diseases have caused huge losses of hybrid grouper culture. However, the immune response of hybrid grouper against viral and bacterial infection remains unexplored. In this study, a total of 9 splenic transcriptomic libraries of hybrid grouper including untreated control, Red-spotted grouper nervous necrosis virus (RGNNV)-challenged, Vibrio alginolyticus-challenged were constructed. A sum of 6024 differentially expressed genes (DEGs) with significant differences were identified, of which the overlapped DEGs between RGNNV and V. alginolyticus challenged were significantly enriched in multiple immune-related pathway including interleukin-17 NOD-like receptor, C-type lectin receptor, chemokine signaling pathway and natural killer cell mediated cytotoxicity, indicating their important roles in immune defense of hybrid grouper against (both) viral and bacterial infection. To the best of our knowledge, this is the first report that investigated the immune response of hybrid grouper following viral and bacterial pathogen stimuli using transcriptomic analysis. These results provide comprehensive understandings of the innate immunity of hybrid grouper and valuable references to develop disease control strategy in grouper aquaculture.

Abstract IA004: Preclinical development of DuoBody®-CD3xB7H4, a novel CD3 bispecific antibody for the treatment of solid cancers

Abstract Several clinical trials are currently underway to investigate the effectiveness of CD3 bispecific antibodies (bsAb) in solid tumors. Given the number of approved CD3 bsAb therapies thus far, the development of these bsAbs appears to be more challenging in solid cancer compared to hematological malignancies. The choice of an appropriate tumor-specific target and CD3 affinity are important considerations for the development of effective and safe CD3 bsAbs. The immune checkpoint protein B7H4 is differentially expressed between normal and tumor tissue, showing high expression in various solid cancers. Therefore, B7H4 provides an attractive target for a CD3 bsAb. We explored the preclinical mechanism of action (MoA) and pharmacodynamic (PD) markers of two B7H4-targeting CD3 bsAbs with different CD3 affinities. By crosslinking CD3 on T cells with B7H4 on tumor cells, CD3xB7H4 bsAbs will induce T-cell mediated cytotoxicity in B7H4-expressing tumor cells, associated with T-cell activation and cytokine production. In comparative preclinical characterization studies in vitro, we showed that both bsAbs induced target-specific, dose-dependent, and complete tumor cell kill in a panel of tumor cell lines expressing varying levels of B7H4, albeit with different potency. Moreover, both molecules demonstrated an acceptable safety profile in cynomolgus monkeys. DuoBody-CD3xB7H4 (GEN1047), the variant with lower CD3 affinity, was selected as clinical candidate, to leverage its observed propensity for lower cytokine production. In a patient-derived xenograft model of ovarian cancer in NCG-HIS mice examined in vivo, DuoBody-CD3xB7H4 showed dose-dependent antitumor activity, which was associated with increased numbers of intratumoral T cells and with peripheral PD biomarkers of T-cell activation and cytokine production. Currently, DuoBody-CD3xB7H4 is being investigated in a first-in-human clinical trial for the treatment of solid tumors known to express B7H4 (NCT05180474), in which the MoA and PD, including biomarkers of response, will be clinically explored. Citation Format: Louise A Koopman, Farshid Alemdehy, Madelon Paauwe, Laura Smits-de Vries, Frosso Karaiskaki, Marcel Brandhorst, Patrick Franken, Theo S Plantinga, Mischa A Houtkamp, Stefanie A.H. de Poot, Esther C.W. Breij. Preclinical development of DuoBody®-CD3xB7H4, a novel CD3 bispecific antibody for the treatment of solid cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA004.

Optimizing Ex-Vivo Expanded NK Cell- Mediated Cellular Cytotoxicity By Obinutuzumab Combined with NKTR-255 in Burkitt Lymphoma (BL)

Background: We have previously reported the success of treating children with Burkitt lymphoma (BL) resulting in a ≥90% long-term EFS utilizing short intensive rituximab containing chemoimmunotherapy (Cairo et al, Blood, 2007; Goldman /Cairo et al, Leukemia, 2012, Goldman /Cairo et al, Br J Haematol., 2014; Goldman /Cairo et al, Leukemia, 2021). However, the prognosis is dismal in children who are refractory or progress after chemoimmunotherapy (Cairo et al Br J Haematol. 2018). The mechanisms associated with this poor prognosis are mainly due to chemoradioimmunotherapy resistance and an immunosuppressive BL microenvironment (TME) (Cairo et al Br J Haematol. 2016; Cairo et al Br J Haematol. 2019) and represent an urgent unmet need. The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully ex-vivo expanded functional and active peripheral blood NK cells (PBNK) with irradiated feeder cells APC (K562-IL21-41BBL) (Chu/Cairo, et al, JITC 2020). We previously demonstrated that obinutuzumab has significantly enhanced ex-vivo expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and NK cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, which may lead to sustained anti-tumor immune response. Objective: To investigate the anti-BL effects of NKTR-255 on the ADCC of ex-vivo expanded NK cells with obinutuzumab against rituximab-resistant BL in vitro and in vivo using human BL xenografted NSG mice. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Lee, et al, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFN-g, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, Front. Immunol, 2022). Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Luciferase expression Raji-4RH cells were xenografted to NSG mice as we previously described (Chu/Cairo, et al, JITC 2021). Results: NKTR-255 significantly enhanced the proliferation of ex-vivo expanded NK cells (p<0.0001) at day 7. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab against rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups. NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN- g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Our in vivo study demonstrated that the combination of NKTR-255, Obinutuzumab and expanded NK cells significantly improved the survival of mice xenografted with Raji-4RH compared to controls (Fig.1). Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with Obinutuzumab against rituximab-resistant BL cells in vitro with enhanced IFN- g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and Obinutuzumab against rituximab-resistant BL cells using humanized NSG models are very promising. Mechanisms studies of BL relapsed from the combination therapy are under investigation. (Supported by HHOW and St Baldrick grants).

G-CSFR Is a Checkpoint of Natural Killer Cells Against Tumor

G-CSFR is a cell surface receptor that can activate cell proliferation and differentiation after binding to granulocyte colony stimulating factor(G-CSF). However, in natural killer (NK) cells, the biological role of G-CSFR has not been fully elucidated. Here we show that granulocyte colony-stimulating factor receptor, that is encoded by the CSF3R gene, is a novel and potent checkpoint in both human and mouse NK cells fighting against tumors. Previous studies have shown that NK cells under resting state with low expression of G-CSFR, but highly expressed in peripheral blood and bone marrow NK cells after G-CSF mobilization. We therefore used G-CSFR blocking agent in vitro to evaluate the effect of G-CSFR on human NK cells proliferation and cytotoxicity capacity. The results showed that G-CSFR agonist significantly down-regulated the proliferation and cytotoxicity of NK cells in vitro and this is independent of G-CSF stimulation. Since NK92 and YT cell lines highly express G-CSFR, we then knocked down G-CSFR in NK92 and YT cells. The results showed that the low expression of G-CSFR enhanced the expression of CD107a in vitro, indicating G-CSFR may be an inhibitory signaling in NK cells. To the further evaluate the specific role of G-CSFR in NK cells and we therefore constructed NK-specific CSF3R deficiency mice (CSF3R △NK/NK; CSF3Rfl/flNCR1cre, thecre group) and the control group ( CSF3Rfl/fl ( CSF3RWT/WT mice, the flox group). We then evaluated the phenotype, differentiation and functional state of NK cells between the two group. NK-specific CSF3R deficiency increased mature NK subsets in mice, and these CSF3R-deficient NK cells exhibited enhanced activation, cytotoxicity and proliferation, which is in accordance with the role in human NK cells. After constructing four tumor-bearing models, namely MC38 (colon tumor), LLC (lung tumor), MLL-AF8(acute myeloid leukemia), B16-F10(melanoma), we found that CSF3R specific depletion in NK cells reduced the tumor burden, prolonged the survival and reduced tumor metastasis in CSF3R△NK/NK mice (Figure 1A-D), indicating CSF3R depletion in NK cells promotes increased ability against tumors. To further evaluate the molecular mechanism of G-CSFR regulation of NK cell function, single cell RNA sequencing on bone marrow cells from both CSF3R△NK/NK mice and CSF3RWT/WT mice and NK cells were extracted for further subpopulation analysis. UMAP analysis revealed the clustering of bone marrow NK cells from both genotypes into ten subsets and on the basis of the key gene expression of NK cell effector molecules, transcriptional factors, maturation markers, and proliferation marker, we identified NK cell maturation program as mBM5+mBM8, mBM2+mBM3+ mBM6 and mBM0, by which immature NK cells should go through to gain optimal effector function, according to this model, CSF3R△NK/NK mice contained more NK cells belonging to the terminally mature mBM0 than CSF3RWT/WT mice. “Cell Killing” and “leukocyte mediated cytotoxicity” pathway was enriched in CSF3R△NK/NK mice, indicating enhanced activation state of NK cells. In conclusion, our results uncover a potent checkpoint in NK cells protecting against tumors, suggesting a promising approach of targeting G-CSFR for NK cell-based immunotherapies.

Improving Anti-Tumor Efficacy of CAR T-Cell Therapy for Acute Myeloid Leukemia (AML): Results from a Multi-Drug Interaction Screening Approach

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and impaired differentiation of myeloid progenitor cells. With a 5-year overall survival of approx. 30%, the prognosis of patients with acute myeloid leukemia remains poor, in particular for those with relapse or refractory disease. The adoptive transfer of T-cells genetically modified to express a chimeric antigen receptor (CAR) have shown impressive remission rates in patients with B-cell lymphoid malignancies. In contrast, CAR T-cell therapy for AML has induced durable remission only in a minority of patients so far, highlighting the need for strategies to improve anti-tumor efficacy and persistence of AML-specific CAR T-cell products. Adapting an established high throughput screening platform, we aimed to identify compounds with the potential to enhance CAR T-cell mediated cytotoxicity through interaction with tumor and/or CAR T-cells. Methods: We established a high throughput platform to determine the cytotoxicity of four different AML-specific CAR T-cell products targeting CD33 (CD33.CD28.41BB.zeta), CD70 (CD27.zeta), CLL1 (CLL1.CD28.zeta) and EMR2 (also known as ADGRE2; EMR2.41BB.zeta) in the presence of 32 compounds at five different concentrations in a 10,000-fold concentration range. CAR T-cells were generated by transduction of activated T-cells derived from peripheral blood mononuclear cells (PBMCs) of three healthy donors with a gamma retroviral vector containing the different CAR constructs. On day 11 after activation, CAR T-cells and non-CAR-transduced control T-cells were incubated with luciferase-expressing target cells at an effector-target ratio of 1:4 in 384-well plates. Viability of target cells cultured with CAR T-cells, control T-cells or in the absence of T-cells was calculated after 24 and 48 hours using a luciferase signal intensity assay. Combinatorial toxicities were calculated with the Bliss Independence model for synergistic effect and the highest single agent model for additive combination effect. Results: In quality analysis, the intensity of luminescence signals in control wells (tumor only, non-CAR-transduced control T-cells) of different plates was similar and principal component analysis (PCA) of samples derived from different days (batches) did not show significant differences ruling out relevant edge or batch effects that could negatively impact the screening results. Of all tested compounds birinapant, a peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of apoptosis protein (IAP) family proteins and the hypomethylating agent (HMA) decitabine showed the strongest improvement of CAR T cell cytotoxicity in an additive but also in a synergistic manner. These effects were evident in CAR T-cell products derived from all different T cell donors without significant differences. Depending on the structural composition of the CAR construct, the strongest drug-induced improvement of CAR T-cell cytotoxicity was seen either after 24 or 48 hours. The third generation CAR T- cell construct (CD33.CD28.41BB.zeta) showed enhanced cytotoxicity already after 24 hours, whereas significant anti-leukemic efficacy of CD27.zeta, CLL1.CD28.zeta and EMR2.41BB.zeta CAR T-cells became evident at the later 48 hours timepoint. Importantly, co-treatment with birinapant and decitabine particularly enhanced the cytotoxicity of EMR2-specific CAR T-cells which exhibited the lowest baseline anti-tumor efficacy compared to all other CAR constructs highlighting the potential of distinct chemical compounds to improve the functionality of AML-specific CAR T-cells. Conclusion: Our data suggest that high throughput drug interaction screens are a reliable approach to investigate the impact of chemical compounds on the functionality of AML-specific CAR T-cell products. HMA and SMAC mimetics are promising candidates that may potentially enhance the cytotoxicity of CAR T-cells. Ongoing studies are evaluating the effect of HMAs and SMAC mimetics on CAR T-cell proliferation and anti-tumor efficacy against AML cell lines and primary AML patient samples upon serial antigen stimulation. Furthermore, in-depth proteogenomic characterization to identify molecular signatures of enhanced functionality are currently being performed.

Anti CD161 Antibody IMT-009 Is a Novel Immunotherapeutic Agent That Effectively Blocks the Inhibitory CLEC2D/CD161 Axis in CLEC2D+ B Cell Hematological Malignancies Reinvigorating T and NK Cell Function Leading to Anti-Tumor Benefit

Background: The CLEC2D/CD161 axis is a ligand-receptor pathway that has been shown to inhibit both T and NK cell function 1,2. CD161 is a C-type lectin-like receptor, which is expressed on NK cells and subsets of both CD4 + and CD8 + T cells. The tumor infiltrating CD161+ T cells have high cytotoxic potential and display an NK cell-like signature 2. Its ligand CLEC2D is expressed on the surface of both malignant cells and immune cells including germinal center B cells, activated T cells and tumor associated macrophages. Owing to its expression in germinal center B cells, CLEC2D is highly expressed in multiple types of B cell lymphomas including Diffuse Large B Cell Lymphoma (DLBCL), Follicular Lymphoma and Burkitt lymphoma 3. Furthermore, subset of infiltrated T cells in these lymphomas are CD161+ making this axis a promising novel target for immunotherapeutic intervention in hematological malignancies (Fig1). Immunitas has developed IMT-009, a monoclonal, Fc silent, fully human IgG1, high affinity antibody to CD161 that selectively blocks its interaction with CLEC2D. We show that IMT-009 effectively reverses CLEC2D-mediated inhibition and restores NK cell and T cell functions, including antigen specific T cell mediated cytotoxicity to lymphoma cell lines. Furthermore, IMT-009 shows monotherapy benefit in reducing tumor burden in a human B cell lymphoma model engrafted in humanized mice in vivo. IMT-009 is currently being evaluated in a Phase 1/2a clinical trial in both solid tumors and heme malignancies. Results: IMT-009 is a highly selective antibody to CD161 that strongly blocks its interaction with CLEC2D. In presence of CLEC2D-expressing target cells, NK cell degranulation, cytokine production and cellular cytotoxicity towards lymphoma tumor targets is highly suppressed which is overcome by treatment with IMT-009. Similarly, IMT-009 reversed CLEC2D-mediated inhibition and restored T cell activity (TCR signaling, cytokine production) in a Jurkat cell reporter system, and enhanced polyfunctionality of primary antigen-specific human T cells shown by secretion of TNF-α, IL-2, and IFN-γ, and direct T cell mediated cytotoxicity. IMT-009 also released CD161-mediated suppression of antigen recall response by PBMC derived effector memory CD161+ CD4+ T cells, resulting in an increased frequency of CD161+ IFN-γ+ cells and an increase in their proliferation. These data together show that IMT-009 is highly effective in overcoming CLEC2D mediated suppression of both T and NK cell functions. To prioritize indications that will likely benefit from IMT-009, we performed IHC for CLEC2D in a diverse set of tumor indications and found that B cell lymphomas of various subtypes have high CLEC2D protein expression by H score. Also, DLBCL was found to have the second highest median RNA expression of CLEC2D in TCGA. Furthermore, analysis of publicly available single-cell RNA-sequencing (scRNA-seq) data from B cell lymphoma including CNS lymphoma revealed CLEC2D to be highly expressed within the B lineage cells including the abundant malignant cell population (Fig2). KLRB1 (gene encoding for CD161) was also found to be expressed on a subset of tumor infiltrating CD4 and CD8 T cells in these datasets (Fig2). To confirm this, we tested CD161 expression in B cell lymphomas by immunofluorescence. Results showed CD161+ cells infiltrated in Hodgkin Lymphoma, and post-R-CHOP treated DLBCL, highlighting the potential sensitivity of these malignancies to IMT-009 treatment. Together, these results indicate that B cell lymphomas may exhibit an immunosuppressive environment mediated by the CLEC2D/CD161 axis. To test the anti-tumor benefit of IMT-009 in a human B cell lymphoma model, we treated CD34+ humanized mice implanted with Raji, a Burkitt lymphoma cell line with IMT-009. The group of mice treated with IMT-009 had a mean tumor growth inhibition of 35% and the anti-tumor response correlated with increased frequency of CD161+ T and NK cells. We further show that IMT-009 can synergize with Rituximab in NK cell mediated killing of B cell lymphoma cell lines in vitro. Conclusion: B cell malignancies tend to respond poorly to anti-PD-1 therapy 4,5. This lack of efficacy could be driven by a highly immunosuppressive environment mediated by the CLEC2D/CD161 axis. Our results support evaluation of IMT-009 as a novel cancer immunotherapy to disrupt CLEC2D/CD161 signaling, with strong therapeutic potential in lymphomas.

Absence of MHC Class I Expression on Target Cells Exacerbates Iron Dependent CD4+ T Cell Mediated Killing

Introduction: Little is known about the target-cell intrinsic features that affect tissue susceptibility to T-cell mediated cell death. The major histocompatibility complex (MHC) Class I presents antigens to CD8+ T cells, while MHC class II presents antigens to CD4+ T cells, and this process is critical for infectious, tumor, auto- and allo -immunity. However, MHC Class I has been suggested to have a non-canonical role iron metabolism, but whether it plays a role in tissue tolerance to CD4+ T cell mediated damage is not known. Herein, we investigated the role of MHC Class I on target cells in CD4+ T-cell mediated damage to intestinal epithelial cells (IECs) utilizing clinically relevant models of T-cell mediated gastrointestinal damage from Graft-versus-Host Disease (GVHD), a major complication of allogeneic stem cell transplantation. Methods: Because MHC class I is expressed on all nucleated cells, to specifically assess the role of MHC Class I on IECs, we generated and utilized intestine-specific MHC Class I knockout mice (B2m∆IEC) as recipients for MHC Class II-disparate bm12–>B2m∆IEC allogeneic stem cell transplant (allo-SCT). We utilized flow cytometry, immunological, molecular and biochemical assays to assess cell death pathways and tissue damage in IECs. We performed mechanistic studies to assess the role of iron and utilized clinically relevant iron chelation treatment to assess survival after treatment. Results: Here, we demonstrate that absence of MHC Class I on target cells exacerbates CD4+ T cell mediated killing of IECs leading to greater mortality in the KO animals than WT recipients (P<0.01) (Fig.1). Compared with wild-type (WT) mice, B2m∆IEC mice, despite an increase in mortality following MHC II disparate allo-SCT, did not demonstrate significant changes in T cell activation, or antigen presentation. In vitro co-culture cell killing assays demonstrate that B2m knockout cells have increased cell death compared with WT cells when co-cultured with activated bm12 CD4+ T cells. Because B2m knockout mice have increased liver iron deposition, we hypothesized that the increase in GI damage might be a consequence of iron related cell death in the IECs in B2m∆IEC mice. We demonstrate that absence of B2m on the intestine is sufficient to drive an increase in tissue iron levels in the liver and intestine. Furthermore, B2m∆IEC allo-recipient mice demonstrated increased intracellular labile iron in IECs. Given the increased mortality in B2m∆IEC allo-recipient mice and the increase in intracellular iron in IECs, we investigated the role of iron-dependent cell death, ferroptosis. We show that IECs from B2m∆IEC allo-recipient mice have increased ferroptosis compared with WT animals (P<0.05). Finally, chelation of iron with administration of clinically utilized iron chelator (Deferasirox) ameliorated excess mortality from GVHD in the B2m∆IEC mice. Conclusion: Absence of MHC Class I expression on target cells exacerbates CD4+ T cell mediated killing by enhancing iron dependent ferroptosis. These data demonstrate that in contrast to immunological paradigm, class I regulates CD4+ T cell mediated cytotoxicity even as it is essential for CD8+ T cell immunity.

Proteomics Landscape and Machine Learning Predictor of Response to Splenectomy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an auto-immune disease characterized by antibody-mediated platelet destruction. Splenectomy remains the effective curative treatment for patients with ITP who failure to respond to TPO-RAs and/or rituximab (Mageau et al, Am J Hematol. 2022). The effectiveness rate of splenectomy for ITP is reported to be roughly 80%, with a 5-year remission rate ranging from 50% to 75% (Chaturvedi et al, Blood. 2018). For subjects presenting with severely low platelet counts or active bleeding, splenectomy represents the primary approach for rapidly increasing platelet levels and potentially saving lives. To achieve personalized and precise treatment, the prediction efficacy on splenectomy remains an urgent problem. Here, we have characterized immune features of ITP patients with splenectomy, and developed a predictive model for assessing the response to splenectomy. Methods Bone marrow plasma samples before splenectomy were collected and analyzed by high-resolution liquid chromatography-tandem mass spectrometry. Weight protein co-expression network analysis (WPCNA) was applied to generate modules from all proteins and mapped them to clinical characteristics. We used least absolute shrinkage and selection operator (Lasso) regression, recursive feature elimination (RFE) for predictors selection. Next, multivariable logistic regression (LR), support vector machine (SVM), and decision tree algorithms were used to develop a predictive model. Results 60 patients with ITP undergoing splenectomy treatment were enrolled into our study, and divided into three groups according to the response [patients with CR (n=30), NR (n=7), and Relapse (n=23)]. A pathway enrichment analysis of WPCNA modules by ClueGO revealed that megakaryocyte differentiation, humoral immune response mediated by circulating immunoglobulin, Fc receptor signaling pathway, and cGMP-PKG signaling pathway were correspondingly enriched in MEtan, MEturquoise, MEblue, and MEpink modules, which were associated with treatments with TPO-RA, IVIG, rhTPO, and post-operation platelet level, respectively. We then performed KEGG pathway enrichment analyses on the different expression proteins (DEPs) to deciphering the immune landscape among each group. For the relapse group, DEPs were primarily enriched in ribosome, spliceosome, neutrophil extracellular trap formation, and necroptosis pathways, comparing with the CR group. For the NR group, most DEPs were enriched in natural killer cell mediated cytotoxicity, NF-kappa B signaling, Fc gamma R-mediated phagocytosis pathways, comparing with the CR group. The models were based on a multi-step predictor pipeline. Inside the pipeline, features were first filtered by lasso regression and RFE algorithm. We identified five proteomic features present in the bone marrow plasm that associated with relapsed to therapy, which included CD40, COL4A3, GALNT6, GSTM3, and UBE2A (Figure). Each ensemble consisted of three algorithms acting in parallel: LR, SVM and decision tree. The AUC values of LR, SVM, and decision tree were 0.962 (95%CI: 0.915-1), 0.957 (95%CI: 0.897-1), and 0.875 (95%CI: 0.782-0.967), respectively. The three algorithms were then tested for performance. The sensitivity, specificity, positive predictive value, and negative predictive value analysis revealed that LR model reached the best performance. Targeting CD40 represents a promising therapeutic approach for enhancing clinical outcomes in patients with ITP who have shown inadequate response following splenectomy. Conclusion Our analysis revealed that ITP patients with splenectomy showed distinct immune characteristics when achieving different response. Protein features extracted from bone marrow plasma can be selected as predictors of response to splenectomy.

3rd-Generation Tyrosine Kinase-Inhibitors and Azacitidine Are Safe and Effective in Myeloid Blast-Phase Chronic Myeloid Leukaemia and Result in a High Proportion of Subjects in 2 nd Chronic Phase Able to Receive a Transplant

Background Myeloidblast phase of chronic myeloid leukemia (CML-MBP) has a terrible prognosis with a haemopoietic cell transplant the only likely cure. Probability of transplant success is markedly increased if someone can be returned to chronic phase. Purposes Study safety and efficacy of olverembatinib or ponatinib and azacitidine (AZA) in this setting. MethodsCML-MBP was diagnosed according to the WHO 2016 criteria. Ponatinib, 45 mg/d or olverembatinib, 30 mg every other d were combined with azacitidine, 75 mg/mE+2/d for 7 d in 28 d cycles. Subjects achieving 2 nd chronic phase received an allotransplant whereas others continued the therapy specified. 2 nd chronic phase was defined as < 10% blood and bone marrow blasts and no extra-medullary leukaemia for ≥ 4 w. Event-free survival (EFS) was defined as interval from therapy start to no response, progression to accelerated or blast phase or CML-related death. CML-related survival was defined as interval from therapy start to death from blast phase and survival, this interval to death from any cause. Haematologic, cytogenetic and molecular responses were monitored. Targeted DNA sequencing and whole RNA sequencing were done before therapy start. “MOVICS” package in R software was used for multi-omics integrated analyses and molecular sub-type classification. Cox regression models were used to identify co-variates associated with outcomes. The study was registered in Chinese Clinical Trial Registry (ChiCTR2200055887). Results 39 consecutive subjects were registered from October, 2020 to July, 2023. 21 were male. Median age was 50 years (Interquartile range [IQR], 33-54 years). 23 received olverembatinib; 16, ponatinib. Median follow-up was 10 months (mo) (IQR, 3-19 mo). 30 (77%) subjects achieved a 2 nd chronic phase after 1 cycle, 11 of whom received a transplant, 8 progressed to blast phase again; and 9 (23%) others had no response. 25 subjects died of progression (N = 19), transplant-related mortality (N = 3), COVID-19 (N = 2) or heart failure (N = 1). Median EFS, CML-related survival and survival were 4 (95% Confidence Interval [CI], 1-6) months, 12 (8-17) months and 11 (6-15) months. Subjects receiving a transplant had better EFS ( p= 0.03), CML-related survival ( p= 0.009) and survival ( p= 0.04) compared with non-transplant subjects ( Figure 1). Most treatment-related severe adverse events were tolerable and manageable including 25 subjects (65%) developing ≥ 4 grade neutropenia with or without thrombocytopenia except only 1 subject developing early death due to heart failure. In addition, 2 subjects experienced COVID-19 and died subsequently. In adjusted Cox regression analyses of genomic (N = 37) and transcriptomic (N = 34) data KRASmutation was significantly-associated with worse EFS ( p= 0.05), CML-related survival ( p< 0.001) and survival ( p= 0.009). TP53mutation was associated with worse CML-related survival ( p= 0.02) and survival ( p= 0.003). PFKFB3::LINC02649 fusion was associated with better EFS ( p= 0.001), CML-related survival ( p= 0.007) and survival ( p= 0.005). Based on these data 34 subjects were divided into 2 clusters. Subjects in cluster 2 (N = 14) had worse EFS ( p= 0.001), CML-related survival ( p= 0.008) and survival ( p= 0.005) compared with those in cluster 1 (N = 20). In differential gene expression analyses 1,564 genes were up-regulated and 1,000 down-regulated in cluster 2 compared with cluster 1. In KEGG analyses up-regulated genes were enriched in signaling pathways including cytokine-cytokine receptor interaction, cell adhesion molecules, natural-killer (NK) cell mediated cytotoxicity, primary immune deficiency and Toll-like receptor. Down-regulated genes were enriched in cell-cycle and DNA replication pathways. Conclusion s Olverembatinib or ponatinib combined with azacitidine is a safe and effective therapy of CML-MBP allowing many subjects to receive a transplant in 2 nd chronic phase. KRAS and TP53 mutations and PFKFB3::LINC02649 fusions had predictive impact on outcomes. Determining whether adding azacitidine to a 3 rd-generation TKI requires a randomized controlled trial.