Leukemia Model Research Articles

Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2.

Leukemia, the most common malignant tumor in childhood, can be categorized into acute leukemia and chronic leukemia. However, the role of FUNDC1 in childhood leukemia (CL) remains unknown. This study aims to investigate the effects of FUNDC1 on patients with CL and its underlying mechanism both in vivo and in vitro. The mRNA expression levels of FUNDC1 were found to be up-regulated in serum samples from CL patients as well as in leukemia cell lines. Furthermore, it was observed that the mRNA expression of FUNDC1 was lower in stage I-II CL patients compared to stage III-IV patients. The up-regulation of FUNDC1 was found to promote leukemia metastasis. Additionally, it was discovered that FUNDC1 up-regulation reduces ferroptosis by inhibiting mitochondrial damage. In a leukemia model, FUNDC1 up-regulation induces the expression of FBXL2. Moreover, FUNDC1 up-regulation reduces FBXL2 ubiquitination, thus maintaining FBXL2 protein expression in leukemia. By inducing FBXL2, FUNDC1 reduces ferroptosis in leukemia through the inhibition of mitochondrial damage. The stability of FUNDC1 is controlled by METTL3 methylation. Overall, this study sheds light on the role of FUNDC1 in CL and provides insights into its underlying mechanisms.

A prognostic model for acute myeloid leukemia based on ferroptosis-related lncRNA and immune infiltration analysis

A prognostic model for acute myeloid leukemia based on ferroptosis-related lncRNA and immune infiltration analysis

Eμ-TCL1 adoptive transfer mouse model of chronic lymphocytic leukemia.

Despite being the most common adult leukemia in the western world, Chronic Lymphocytic Leukemia (CLL) remains a life-threatening and incurable disease. Efforts to develop new treatments are highly dependent on the availability of appropriate mouse models for pre-clinical testing. The Eμ-TCL1 mouse model is the most established pre-clinical approach to study CLL pathobiology and response to treatment, backed by numerous studies highlighting its resemblance to the most aggressive form of this malignancy. In contrast to the transgenic Eμ-TCL1 model, employing the adoptive transfer of Eμ-TCL1-derived splenocytes in immunocompetent C57BL/6 mice results in a comparably rapid (e.g., leukemic development within weeks compared to months in the transgenic model) and reliable model mimicking CLL. In this chapter, we would like to provide readers with a thoroughly optimized, detailed, and comprehensive protocol to use the adoptive transfer Eμ-TCL1 model in their research.

Influence of the age structure on the stability in a tumor-immune model for chronic myeloid leukemia

In this paper a model of tumor-immune response for chronic myeloid leukemia (CML) is proposed and analyzed. It is based on the ordinary differential equations’ models (ODE) studied in A. Besse, et al., Bull. Math. Biol. (2017) 1—27, G.D. Clapp, et al., Cancer Res. 75 (2015) 4053-4062. The proliferation of cells, their differentiation in the bone marrow and the interactions of leukemic and immune cells are described. The model is based on a non-monotonic immune response. At low levels immune response increases with the tumor load, whereas at high levels tumor is suppressing the effect of the immune system (immunosuppression). We consider that the age of cells is described by a continuous variable which we use to structure the system and obtain a partial differential equations’ model (PDEs). We analyze the stability of the equilibrium points of the model and compare it to the case of A. Besse, et al., Bull. Math. Biol. (2017) 1—27 where age was described as a discrete state. In particular, an equilibrium point describing remission, induced by a control of the immune system, is shown to be unstable in certain situations for the PDE model, whereas in A. Besse, et al., Bull. Math. Biol. (2017) 1-27 it was systematically stable.

Invivo and invitro evaluation of antitumor effects of iron oxide and folate core shell-iron oxide nanoparticles.

Nanoparticles are considered viable options in the treatment of cancer. This study was conducted to investigate the effect of magnetite nanoparticles (MNPs) and magnetite folate core shell (MFCS) on leukemic and hepatocarcinoma cell cultures as well as their effect on the animal model of acute myelocytic leukemia (AML). Through current study nanoparticles were synthesized, characterized by various techniques, and their properties were studied to confirm their nanostructure. Invivo study, nanoparticles were evaluated to inspect their cytotoxic activity against SNU-182 (human hepatocellular carcinoma), K562 (human leukemia), and THLE2 (human normal epithelial liver) cells via MTT test. Apoptotic signaling proteins Bcl-2 and Caspase-3 expression were inspected through RT-PCR method. A cytotoxic effect of MNPs and MFCS was detected in previous cell cultures. Moreover, the apoptosis was identified through significant up-regulation of caspase-3, with Bcl-2 down-regulation. Invitro study, AML was induced in rats by N-methyl-N-nitrosourea followed by oral treatment with MNPS and MFCS. Biochemical indices such as aspartate and alanine amino transferases, and lactate dehydrogenase activities, uric acid, complete blood count, and Beta -2-microglubulin were assessed in serum. Immunophenotyping for CD34 and CD38 detection was performed. Liver, kidney, and bone marrow were microscopically examined. Bcl-2 promoter methylation, and mRNA levels were examined. Although, both MNPs and MFCS depict amelioration in biochemical parameters, MFCS alleviated them toward normal control. Anticancer activity of MNPs and MFCS was approved especially for AML. Whenever, administration of MFCS was more effective than MNPs. The present work is one of few studies used MFCS as anticancer agent.

Artificial intelligence-based prediction models for acute myeloid leukemia using real-life data: A DATAML registry study

We designed artificial intelligence-based prediction models (AIPM) using 52 diagnostic variables from 3687 patients included in the DATAML registry treated with intensive chemotherapy (IC, N = 3030) or azacitidine (AZA, N = 657) for an acute myeloid leukemia (AML). A neural network called multilayer perceptron (MLP) achieved a prediction accuracy for overall survival (OS) of 68.5% and 62.1% in the IC and AZA cohorts, respectively. The Boruta algorithm could select the most important variables for prediction without decreasing accuracy. Thirteen features were retained with this algorithm in the IC cohort: age, cytogenetic risk, white blood cells count, LDH, platelet count, albumin, MPO expression, mean corpuscular volume, CD117 expression, NPM1 mutation, AML status (de novo or secondary), multilineage dysplasia and ASXL1 mutation; and 7 variables in the AZA cohort: blood blasts, serum ferritin, CD56, LDH, hemoglobin, CD13 and disseminated intravascular coagulation (DIC). We believe that AIPM could help hematologists to deal with the huge amount of data available at diagnosis, enabling them to have an OS estimation and guide their treatment choice. Our registry-based AIPM could offer a large real-life dataset with original and exhaustive features and select a low number of diagnostic features with an equivalent accuracy of prediction, more appropriate to routine practice.

An ensemble-acute lymphoblastic leukemia model for acute lymphoblastic leukemia image classification.

The timely diagnosis of acute lymphoblastic leukemia (ALL) is of paramount importance for enhancing the treatment efficacy and the survival rates of patients. In this study, we seek to introduce an ensemble-ALL model for the image classification of ALL, with the goal of enhancing early diagnostic capabilities and streamlining the diagnostic and treatment processes for medical practitioners. In this study, a publicly available dataset is partitioned into training, validation, and test sets. A diverse set of convolutional neural networks, including InceptionV3, EfficientNetB4, ResNet50, CONV_POOL-CNN, ALL-CNN, Network in Network, and AlexNet, are employed for training. The top-performing four individual models are meticulously chosen and integrated with the squeeze-and-excitation (SE) module. Furthermore, the two most effective SE-embedded models are harmoniously combined to create the proposed ensemble-ALL model. This model leverages the Bayesian optimization algorithm to enhance its performance. The proposed ensemble-ALL model attains remarkable accuracy, precision, recall, F1-score, and kappa scores, registering at 96.26, 96.26, 96.26, 96.25, and 91.36%, respectively. These results surpass the benchmarks set by state-of-the-art studies in the realm of ALL image classification. This model represents a valuable contribution to the field of medical image recognition, particularly in the diagnosis of acute lymphoblastic leukemia, and it offers the potential to enhance the efficiency and accuracy of medical professionals in the diagnostic and treatment processes.

Enhancing CAR-T cells: unleashing lasting impact potential with phytohemagglutinin activation in in vivo leukemia model.

Chimeric antigen receptor T (CAR-T) cell therapy holds great promise as an innovative immunotherapeutic approach for cancer treatment. To optimize the production and application of CAR-T cells, we evaluated the in vivo stability and efficacy capacities of CAR-T cells developed under different conditions. In this study, CAR-T cells were activated using Phytohemagglutinin (PHA) or anti-CD3&anti-CD28 and were compared in an in vivo CD19+B-cell cancer model in mouse groups. Our results demonstrated that CAR-T cells activated with PHA exhibited higher stability and anti-cancer efficacy compared to those activated with anti-CD3&anti-CD28. Specifically, CAR19BB-T cells activated with PHA exhibited continuous proliferation and long-term persistence without compromising their anti-cancer efficacy. Kaplan-Meier survival analysis revealed prolonged overall survival in the CAR-T cell-treated groups compared to the only tumor group. Furthermore, specific LTR-targeted RT-PCR analysis confirmed the presence of CAR-T cells in the treated groups, with significantly higher levels observed in the CAR19BB-T (PHA) group compared to other groups. Histopathological analysis of spleen, kidney, and liver tissue sections indicated reduced inflammation and improved tissue integrity in the CAR-T cell-treated groups. Our findings highlight the potential benefits of using PHA as a co-stimulatory method for CAR-T cell production, offering a promising strategy to enhance their stability and persistence. These results provide valuable insights for the development of more effective and enduring immunotherapeutic approaches for cancer treatment. CAR-T cells activated with PHA may offer a compelling therapeutic option for advancing cancer immunotherapy in clinical applications.

Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.

Interleukin-9 production by type 2 innate lymphoid cells induces Paneth cell metaplasia and small intestinal remodeling

Paneth cell metaplasia (PCM) typically arises in pre-existing gastrointestinal (GI) diseases; however, the mechanistic pathway that induces metaplasia and whether PCM is initiated exclusively by disorders intrinsic to the GI tract is not well known. Here, we describe the development of PCM in a murine model of chronic myelogenous leukemia (CML) that is driven by an inducible bcr-abl oncogene. Mechanistically, CML induces a proinflammatory state within the GI tract that results in the production of epithelial-derived IL-33. The binding of IL-33 to the decoy receptor ST2 leads to IL-9 production by type 2 innate lymphoid cells (ILC2) which is directly responsible for the induction of PCM in the colon and tissue remodeling in the small intestines, characterized by goblet and tuft cell hyperplasia along with expansion of mucosal mast cells. Thus, we demonstrate that an extra-intestinal disease can trigger an ILC2/IL-9 immune circuit, which induces PCM and regulates epithelial cell fate decisions in the GI tract.

Abstract C095: The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia

Abstract Introduction: Over 70% of infants with acute lymphoblastic leukemia (ALL) present with mixed lineage leukemia (MLL1/KMT2A) gene rearrangements (MLL-r ALL), which is associated with poor prognosis. MLL-r encodes oncogenic fusion proteins that cause aberrant chromatin histone methylation and leukemic transformation of hematopoietic progenitors. A characteristic of MLL-r leukemia is high expression of the FLT3 gene. Menin is critical for leukemic transformation by MLL fusion proteins. VTP-50469, the preclinical precursor of revumenib (SNDX-5613), is a potent and selective small molecule inhibitor of the Menin-MLL interaction that has shown impressive single-agent activity in preclinical models of MLL-r acute leukemia. Revumenib is currently undergoing clinical evaluation, therefore it was of interest to assess the in vivo activity of VTP-50469 in combination with established drugs against MLL-r ALL patient-derived xenografts (PDXs). Methods: VTP-50469 was tested as a single agent against a panel of 7 pediatric MLL-r ALL PDXs in immune-deficient (NSG) mice (120 mg/kg twice daily x 28 days via oral gavage). Combination studies used VTP-50469 formulated in chow (0.1%) available ad libitum for 28 days in combination with either an induction-type regimen of vincristine (0.15 mg/kg once weekly x 4 weeks), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off x 4 weeks, VXL) all administered intraperitoneally, or the FLT3 inhibitor gilteritinib (Xospata, 30 mg/kg x 28 days via oral gavage). Events were defined as >25% huCD45+ in the peripheral blood. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C measurements and stringent objective response criteria (progressive disease, PD; complete response, CR; maintained CR, MCR), and disease infiltration in the spleen and femoral bone marrow (BM) at 28 days post-treatment initiation. Results: Single-agent VTP-50469 elicited MCRs in 6/7 PDXs and significantly (P<0.0001) reduced leukemia infiltration of the spleen (7/7 PDXs) and BM (6/7 PDXs) compared to vehicle control mice. When administered in chow, VTP-50469 elicited a CR in the MLL-7 PDX (T-C 48.2 days, T/C 8.1) and a PD in MLL-8 (T-C 23.0 days, T/C 4.9). VTP-50469 combined with VXL elicited MCRs in both PDXs (MLL-7 T-C 77.0 days, T/C 12.3; MLL-8 T-C 49.1 days, T/C 9.3) and significantly delayed leukemia progression compared with VTP-50469 or VXL alone (P=0.0013). Moreover, VTP-50469 combined with VXL almost completely eradicated leukemia infiltration of spleens and BMs by both PDXs. VTP-50469 combined with gilteritinib did not result in therapeutic enhancement compared to each single agent arm in either PDX. Conclusions: The addition of VTP-50469 to a standard-of-care induction-type regimen (VXL) resulted in therapeutic enhancement against two infant MLL-r ALL PDXs, while VTP-50469 plus gilteritinib was substantially less effective. These results support further clinical evaluation of revumenib in combination with standard-of-care therapy in MLL-r leukemia. Citation Format: Richard B Lock, Kathryn Evans, Sophia Gawan-Taylor, Ben Watts, Timothy Stearns, Eric J Earley, Emily L Jocoy, Carol J Bult, Beverly A Teicher, Gerard M McGeehan, Malcolm A Smith. The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia [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 C095.

Abstract A053: The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML)

Abstract The BRG/BRM associated factor (BAF) complex is a chromatin remodeler critical for maintenance of cell viability in many cancers. This includes acute myeloid leukemia (AML), where BAF maintains the stem-like transcriptional state of blast cells. FHD-286 is a dual inhibitor of the BAF ATPase subunits BRG1 and BRM and is currently being investigated in relapsed or refractory AML and myelodysplastic syndrome. We have shown previously that FHD-286 induces the expression of the myeloid differentiation marker CD11b in preclinical models after treatment with sub-cytoreductive doses at long timepoints. Here we further characterized the differentiation phenotype induced by low-dose FHD-286 in AML cell lines through RNA-seq and a genome-wide CRISPR-Cas9 knockout screen. These datasets and mechanistic follow-up studies suggested AML cell lines treated with FHD-286 can functionally differentiate to gain the ability to produce superoxide anion and perform phagocytosis. We also found that FHD-286 treatment disrupts mRNA splicing and that this is a likely contributor to AML cell growth defects induced by BRG1/BRM inhibition. This study suggests multiple mechanisms by which FHD-286 is able to disrupt the stem-like transcriptional state of AML blasts to cause differentiation and ultimately cell death. Citation Format: Ashley K. Gartin, David N. Mayhew, David L. Lahr, GiNell Elliott, Heena Gandevia, Molly M. Wilson, Richard Centore, Mike Collins, Astrid Thomsen, Jessica Piel, Gabriel J. Sandoval, Martin Hentemann. The dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 induces functional differentiation and splicing defects in preclinical models of acute myeloid leukemia (AML) [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 A053.

Abstract A049: Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia (AML) is a complex disease with multiple sub-types, each characterized by unique clinical and molecular features, driving the need to develop targeted therapies which exploit specific vulnerabilities. Bromodomain-containing protein 9 (BRD9) is a component of the ncBAF chromatin remodeling complex, and has been recently indicated as a strong dependency in AML (Weisberg et al 2022). It has been shown that inhibitors of BRD9 induce growth inhibition and expression of apoptotic makers in AML cell lines (Hohmann et al 2016; Zhou et al 2021). It has also been reported that BRD9 is critical to AML cell survival through the maintenance of STAT5 signaling (Del Gaudio et al 2019). Herein, we profile the in vitro anti-proliferative effects of FHD-609, a potent and selective degrader of BRD9, in a panel of 40 AML cell lines representative of a broad range of AML sub-types. We observed that FHD-609 was effective at inhibiting the growth of a subset of AML cell lines, consistent with previous reports in the literature. We further investigated whether treatment of AML cell lines with FHD-609 would induce changes in cell cycle, and found an increase in G1 in the same subset of cell lines that showed cell growth inhibition. Furthermore, treatment with FHD-609 induced apoptosis in these cell lines. To identify the mechanisms of action of FHD-609 in AML, as well as potential predictive biomarkers for AML sensitivity to FHD-609, we performed a range of mechanistic studies including ATACseq, ChIPseq and RNAseq in AML cells treated with FHD-609. We observed significant changes in the chromatin landscape in sensitive AML cell lines, and negligible changes in insensitive cell lines. Further bioinformatics analysis identified a possible biomarker strategy that correlates with the sensitivity of AML cell lines to FHD-609 in vitro. To confirm whether this biomarker strategy would successfully predict response in vivo, we dosed AML CDX and PDX models with FHD-609 based on the biomarker selection strategy. We observed a strong anti-tumor effect in AML CDX models and significantly extended survival of the mice in the AML PDX models. In summary, we have shown that FHD-609 demonstrates strong anti-tumor efficacy in a subtype of AML and have identified a possible biomarker strategy to predict response. Citation Format: Claudia Dominici, David Mayhew, Ammar Adam, Flore Uzan, Victoria Garbitt-Amaral, Oliver Mikse, Brandon Antonakos, Hafiz Ahmad, Salonee Parikh, Mei Yun Lin, Gabriel Sandoval, David Lahr, Hsin-Jung Wu, Mengni Xu, Sean Brennan, Luis M. M. Soares, Jordana Muwanguzi, Huawei Chen, Zhaoxia Yang, Jason T Lowe, Matt Netherton, Laura Zawadzke, Johannes Voigt, Liyue Huang, Sabine Ruppel, Ho Man Chan, Ryan Kruger, David S Milan, Scott Innis, Qianhe Zhou, Steven F Bellon. Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML) [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 A049.

Abstract C077: STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment

Abstract Background METTL3 is the RNA methyltransferase responsible for the deposition of N-6-methyladenosine modification (m6A) on mRNA, to regulate its stability, splicing and protein translation. Small molecule inhibitors of METTL3 catalytic activity have previously demonstrated anti-tumor efficacy in pre-clinical models of acute myeloid leukemia (AML) and solid tumors. Inhibition of METTL3 was shown to induce a cell-intrinsic innate immune response through activation of the double strand RNA (dsRNA) sensing machinery and interferon (IFN) signalling. Here we present new pre-clinical data, investigating the combination of METTL3 inhibitor (METTL3i) with DNA damaging therapies. We show that METTL3 inhibition profoundly changes the tumor microenvironment (TME), and we further demonstrate the generation of durable immune memory in mice treated with METTL3i, with or without anti-PD1 therapy. Materials & Methods The orally bioavailable small molecule clinical-stage METTL3i STC-15, or the equipotent tool compound STM3675 were used in these studies. To characterise transcriptomic changes following METTL3 inhibition, RNA sequencing was performed. Induction of specific genes was validated by Western Blot, and cytokine secretion assessed by ELISA. In-vitro cell viability was assessed by Cell TiterGlo (Promega). In vivo efficacy studies used subcutaneous MC38 and GL261 syngeneic tumor models, and characterisation of the TME was performed by flow cytometry. Results Treatment with METTL3i sensitized cells to DNA damaging agents such as doxorubicin. On the molecular level, further enhancement of the IFN pathway by the combination treatment was observed. Moreover, combination of METTL3i with radiation treatment (RT) in vivo in the MC38 colorectal syngeneic mouse model demonstrated a clear benefit of the combination over each of the single treatments. METTL3i alone or in combination with RT led to a major TME remodulation, reducing the number of Tregs, M2 macrophages and neutrophils, and increasing the number of M1 macrophages and CD8+ T-cells in tumors. The GL261 syngeneic glioma model is exquisitely sensitive to anti-PD1 treatment. Similarly, METTL3i treatment led to multiple complete regressions, and the combination of METTL3i and anti-PD1 further accelerated the occurrence of regressions. Following a 3-weeks’ observation period, tumor free mice were re-inoculated with GL261 cells. All tumor-free mice remained tumor free after the rechallenge. Conclusions In pre-clinical cancer models, treatment with METTL3i such as STC-15 resulted in activation of innate immune pathways and subsequent enhancement of cytotoxic T-cell activity. In-vivo, STC-15 had a profound effect on the TME, shifting it from an immunosuppressive, pro-tumorigenic state to an immunostimulatory, anti-tumor state. In combination with anti-PD1, METTL3 inhibitors generate a durable anti-tumor response and lasting immune memory. A First-in-Human dose escalating study evaluating STC-15 monotherapy in patients with solid tumors is ongoing (NCT05584111). Citation Format: Yaara Ofir-Rosenfeld, Lina Vasiliauskaitė, Joanna Obacz, Georgia Tsagkogeorga, Josefin-Beate Holz, Jerry McMahon, Oliver Rausch. STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment [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 C077.

Establishment and validation of a prognostic immune-related lncRNA risk model for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a cancer arising in the bone marrow and is the most common type of adult leukemia. AML has a poor prognosis, and currently, its prognosis evaluation does not include immune status assessment. This study established an immune-related long non-coding RNA (lncRNA) prognostic risk model for AML based on immune lncRNAs screening. To construct training and validation cohorts, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public databases were accessed to obtain gene expression profiles and clinical data. The correlation between lncRNAs and immunity genes was analyzed using the "limma" package, and the immune-related lncRNAs were obtained. Through least absolute shrinkage and selection operator regression, a prognostic model was established with immune-related lncRNAs. Using the median risk score, patients were divided into high- and low-risk groups. The Kaplan-Meier method was used for survival analysis, whereas the accuracy of the risk model was evaluated using time-dependent receiver operating characteristic curves, risk score distribution, survival status, and risk heat maps. We utilized univariate and multivariate Cox regression to examine the association between risk score and clinical variables and AML survival and prognosis. In the immune-related lncRNA prognostic risk model, the prognosis was better for low-risk than for high-risk patients, indicating risk score of this model as an independent indicator of prognosis. The area under the curve value for 1-, 3-, and 5-year survival of TCGA patients was 0.817, 0.859, and 0.909, respectively, whereas that of GEO patients (of dataset GPL96-GSE37642) was 0.603, 0.652, and 0.624, respectively. Gene set enrichment analysis revealed the enrichment of multiple pathways, such as antigen processing, B-cell receptor signaling pathway, natural killer cell-mediated cytotoxicity, and chemokines, in high-risk patients. In this study, immune-related lncRNA prognostic risk models effectively predicted AML survival and provided potential treatment targets.

Picalm/MLLT10 Expression in Zebrafish Is Associated with an Increase of Hematopoietic Stem Cells and Leads to Leukemia

The PICALM/ MLLT10 (C/A)fusion gene is the result of the (10;11)(p13;q14) translocation, and is found in about 1% of all AML patients, 30% of T-cell ALL with a TCR γ/∂ rearrangement and in some patients with lymphoma. AML patients with a t(10;11)(p13;q14) have poor long-term survival. Mouse models of t(10;11)(p13;q14) show that C/A and the C/A minimal fusion (CA/MF) are strong oncogenic drivers, and that C/A must be expressed in the hematopoietic stem cells (HSCs) to cause leukemia. Patient samples and mouse models of C/A leukemia show that these leukemias harbor additional, cooperating mutations. Zebrafish (ZF) have become an attractive animal model for studying hematological disorders. ZF can be easily genetically modified and kept in large numbers. Although T cell ALL models have been successful in ZF, AML models have not been as successful. Most ZF AML models fail to recapitulate the phenotype, especially transplantability. Here we report a C/A transgenic zebrafish AML model (R:CA/MF). We used the murine Runx1 promoter to direct CA/MF expression to zebrafish HSCs using Tol2-mediated transgenesis. We detected the expression of the CA/MF transcript using RT-PCR in 3 dpf (days post fertilisation) heart marker positive transgenic F0 embryos and the expression of FLAG-tagged CA/MF protein with whole mount in situ immuno histochemistry in stable transgenic R:CA/MF embryos at 3 dpf. We performed whole mount in situ hybridisation (WMISH) and qPCR for hematopoietic genes to examine the impact of CA/MF on zebrafish hematopoiesis. Based on qPCR analysis, we observed that the expression of pu1 (p value <0.0120) and gata1 (p value <0.0004) was significantly decreased in 3 dpf R:CA/MF embryos compared to WT, whereas the expression of mpx at 2 dpf (p value <0.0132) and 3 dpf (p value <0.0378) was significantly increased. Our WMISH analysis showed that the expression of lyz was significantly decreased in R:CA/MF compared to WT embryos at 2 dpf. To investigate the effect of CA/MF expression on HSCs, we injected the R:CA/MF construct into fertilised oocytes of R:GFP;kdrl:RFP fish (R:GFP marks HSCs and Kdrl:RFP marks blood vessels). The results from intravital confocal microscopy showed that the 3 dpf R:GFP larvae that had been injected with the R:CA/MF vector had about a 2.5 fold increase (p value <0.0001) of the GFP expressing cells (HSCs) in both the caudal hematopoietic tissue and circulation compared to control (R:Gal4) and un-injected larvae. These results were also confirmed by flow cytometry. CA/MF transgenic zebrafish developed an aggressive hematological malignancy with a latency of about 1 year and 60 to 70% penetrance. 68% of F1 R:CA/MF (15/22), 64% of F2 R:CA/MF (45/70), 62% of F3 R:CA/MF (22/35) and 62% of F4 R:CA/MF (25/40) adult ZF were euthanised or found dead. The median survival for F1 CA/MF transgenic ZF was 65.6 weeks and 69, 78.4, and 79.1 weeks for F2, F3, and F4, respectively. Histology analysis of R:CA/MF leukemic fish showed hypercellularity in kidney, spleen, heart, gills and eyes. Myeloperoxidase (MPO) staining showed that the number of MPO positive cells in kidney marrow was about 50% higher (p value <0.0001) in R:CA/MF compared to WT. Peripheral blood film analysis of R:CA/MF showed the presence of blast-like cells. In addition, R:CA/MF malignancies were associated with an expansion of either the precursor compartment (N=20/31), the myeloid compartment (N=8/31) or the lymphoid compartment (N=3/31) on forward/side scatter flow cytometry analysis. Transplantation assays showed that R:CA/MF leukemias were serially transplantable. The latency to leukemia development was about six weeks in the first, second, and third recipient fish, which was much shorter than the latency for the primary R:CA/MF leukemias. Our preliminary whole-exome sequencing (WES) analysis identified somatically mutated genes in CA/MF zebrafish leukemia with some of these genes having human orthologues that are known to be somatically mutated in human leukemia suggesting that these are cooperating driver mutations. Preliminary RNA-Seq experiments revealed that genes in the KRAS and MTOR pathways had increased expression in the CA/MF ZF leukemia. R:CA/MF leukemia model is a valuable model for understanding the molecular mechanisms of malignant cell transformation of C/A. It allows us to investigate the effects of C/A on HSCs in vivo and to study the fully developed leukemia via serial transplantation, WES, and RNA-Seq.

RSK1 Is an Exploitable Dependency in Myeloid Malignancies

Myeloid malignancies harbor distinct molecular drivers but share convergence of oncogenic signaling pathways and propagation by ripe pro-inflammatory niches. To delineate these hallmarks across the spectrum of myeloid disease states, we established the largest comprehensive atlas of myeloproliferative neoplasms (MPN) and secondary acute myeloid leukemia (sAML) through RNA-sequencing and mass cytometry (CyTOF) of 370 primary samples encompassing CD34+ hematopoietic stem/progenitor cells and CD14+ monocytes, revealing aberrant of PI3K/AKT/mTOR signalling and NFκB-mediated hyper-inflammation. As a central mediator of these hyperactive signaling pathways, ribosomal S6 kinases (RSKs) represent potentially attractive novel therapeutic targets warranting further evaluation. Using both genetic approaches and a first-in-class oral RSK inhibitor, PMD-026, that is currently being evaluated in phase 1/1b clinical trials (NCT04115306) in metastatic and triple negative breast cancer, we found that RSK1 ( RPS6KA1) inhibition potently induced apoptosis and G2/M arrest in leukemia models in conjunction with suppression of PI3K/AKT/mTOR and NFκB signaling. Further NFκB cascade profiling following RSK1 perturbation revealed reduced phosphorylation of IKKα/β and p65/RELA and prevention of p65/RELA nuclear translocation. Using RNA-seq, ATAC-seq and H3K27ac CUT&Tag, we found that PMD-026 treatment altered chromatin accessibility and inhibited NFKB1, TNF, and cell cycle regulators CCNA1/2 and CDK1/2. In cytokine CyTOF, PMD-026 nearly abrogated all induction of inflammatory cytokines including TNF, IL-6, IL-8, CCL3, and CCL4 in primary MPN monocytes to levels substantially below basal conditions. We further extended our findings to de novo AML harboring FLT3 internal tandem duplication ( FLT3-ITD), where FLT3-ITD patients exhibited elevated RPS6KA1 expression and those who developed resistance to gilteritinib demonstrated progressively increased RPS6KA1 expression. FLT3-ITD cells exhibited preferentially sensitivity to RSK1 inhibition, where we uncovered bi-directional regulation. Through cycloheximide, MG-132, and ubiquitination assays, we found that RSK1 regulates FLT3-ITD activity and protein stability through deubiqutinase USP1, and where either RPS6KA1 or USP1 inhibition resulted in concomitant downregulation of FLT3 protein and cell lethality. Multivariate analysis of the TCGA and BeatAML cohorts (n= 568 patients) revealed high median USP1 or RPS6KA1 expression to be significantly associated with poor survival, thus defining a RSK1-USP1-FLT3 axis with prognostic relevance. Across in vivo models, PMD-026 ameliorated MPN disease features driven by MPL W515L including leukocytosis, splenomegaly, and bone marrow fibrosis, while prolonging survival, in conjunction with suppression of inflammatory cytokines including TNF, IL-6 and IL-1b. We then evaluated PMD-026 across five patient-derived xenograft (PDX) models spanning the spectrum of myeloid malignancies including myelofibrosis (MF), sAML, and chronic myelomonocytic leukemia (CMML) encompassing various driver and multiple high-risk mutations. Treatment in MF PDX mice decelerated disease progression, extended survival, and reduced splenomegaly, while in CMML/sAML PDXs, PMD-026 led to a 67 to 96 percent reduction in human CD45+ cells engraftment. Finally, PMD-026 treatment in both mice xenografted with MV4-11 cells and syngeneic Flt3ITDTet2KO mice significantly reduced leukemic cell engraftment and prolonged survival. These data indicate potent efficacy of RSK inhibition across diverse models of chronic and acute myeloid malignancies. Lastly, we investigated the impact of PMD-026 on hematological parameters from 41 breast cancer patients enrolled in the phase 1/1b clinical trial. Overall, there was no sustained suppressive effect on hematological parameters and there were no hematological PMD-026-related toxicities meeting criteria for dose modification during the trial period. These clinical data demonstrate that PMD-026 did not cause myelosuppression. Our findings uncover a novel therapeutic avenue for a conserved RSK1 dependency across chronic and acute myeloid neoplasms. The potent and consistent disease-ameliorating effects across numerous leukemia mouse models demonstrates promise for repurposing PMD-026 for the treatment of myeloid malignancies.

SHP2 Inhibition Enhances JAK2 Inhibitor Therapy in Preclinical Models of Myeloproliferative Neoplasms

The classic myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are driven by aberrant activation of the JAK2 kinase, primarily by mutations in 3 different genes, JAK2, MPL, and CALR. JAK2 inhibitor therapy is generally effective at reducing MPN patient constitutional symptoms and thus can improve quality of life but has minimal effect on reducing mutant driver allele burden and does not readily alter the course of disease, reflecting the ability of MPN-driving cells to persist during therapy. Thus, the ability of current anti-JAK2 therapies to significantly modify underlying disease biology is limited. Long-term studies of the JAK2 inhibitor ruxolitinib in myelofibrosis patients have, however, identified survival benefits, yet this may be due to anti-inflammatory and spleen reducing impacts. Ultimately, the inability of ruxolitinib and other approved JAK2 inhibitors to effectively antagonize allele burden indicates alternative therapeutic approaches are needed to overcome the persistent survival of MPN-driving cells during JAK2 inhibitor therapy. Efforts to identify mechanisms that can thwart the effects of JAK2 inhibition in MPN cells have highlighted activation of the RAS-ERK pathway, including mechanisms of RAS-ERK signaling that are independent of JAK2. ERK or MEK inhibition have been shown to enhance the activity of ruxolitinib in MPN mouse models, but monotherapeutic inhibition of these kinases displayed limited efficacy. The SHP2 (Src homology region 2 domain-containing phosphatase-2) tyrosine phosphatase mediates RAS activation and subsequent MEK/ERK signaling downstream of cytokine and growth factor receptors. SHP2 plays a role in oncogenic signaling and in resistance to kinase targeted therapies, exemplified in both solid tumor and leukemia models. Given the evidence that activation of RAS-ERK signaling can antagonize the effects of JAK2 inhibition and the fact that SHP2 mediates RAS activation via multiple mechanisms downstream of numerous growth factor and cytokine receptors, we hypothesized that SHP2 may contribute to the survival of MPN cells during JAK2 inhibitor treatment and may provide a therapeutic target to improve current anti-JAK2 therapies. Using preclinical MPN models, we aimed to block RAS activation by inhibiting SHP2 to assess the potential SHP2 inhibition may have as a monotherapy or in combination with JAK2 inhibition. We found that the SHP2 inhibitors RMC-4550 and SHP099 enhanced growth inhibition of MPN model cell lines (e.g., SET2 and UKE1) in combination with ruxolitinib, effectively preventing ruxolitinib persistent growth. Concomitant treatment with both inhibitors led to enhanced inhibition of RAS-GTP levels and ERK activation and increased apoptosis compared to single agents. SHP2 inhibition antagonized the neoplastic growth of primary hematopoietic progenitor cells from MPN patients and synergized with ruxolitinib in all patient samples assessed. In an MPN mouse model driven by MPL-W515L, RMC-4550 antagonized MPN phenotypes, including leukocytosis, hepatosplenomegaly, and allele burden. RMC-4550 treatment diminished levels of a subset of pro-inflammatory cytokines, including IL-1β, CXCL9, and TNFα, among others. The combination of RMC-4550 with ruxolitinib not only suppressed advancing leukocytosis but led to white blood cell levels lower than at treatment initiation (Panel A) and suppressed thrombocytosis compared to ruxolitinib alone. The combination also led to enhanced inhibition of hepatomegaly, extramedullary hyperplasia, megakaryocyte hyperplasia, and allele burden, and a trend toward improved fibrosis. The SHP2 inhibitor RMC-4550 enhanced the survival of mice (following termination of treatment analyses) as both a monotherapy (p < 0.0001 compared to vehicle) and in combination with ruxolitinib (p < 0.01 compared to ruxolitinib monotherapy). Importantly, the combination of SHP2 inhibition using RMC-4550 with JAK2 inhibition using ruxolitinib for 4 weeks in wildtype mice was well tolerated with respect to hematologic parameters and exemplified by no effect on body weight (Panel B). Given SHP2 inhibitors are already undergoing clinical evaluation in patients with solid tumors, our findings suggest that SHP2 is a therapeutic target with potential to be rapidly translated to clinical assessment for MPN patients.

Long-Acting E. coli-Derived Asparaginase Potentiates the Anti-Leukemic Effect of BCL2 Inhibition, but Not MCL1 Inhibition, in Preclinical Models of Acute Myeloid Leukemia

Background: Asparaginases are enzymes that deplete circulating asparagine and glutamine from plasma and can therefore be used therapeutically for cancers that are sensitive to amino acid depletion. Commercially available asparaginases are isolated fromeither E. coli or Erwinia chrysanthemi (also called crisantaspase), the latter having a higher glutaminase activity. Our previous studies have shown that pegcrisantaspase (PegC), a long-acting pegylated crisantaspase, synergizes with the BCL2 inhibitor, Venetoclax (Ven), to kill several AML cell lines and patient-derived primary AML cells with complex karyotype in vitro and in vivo in a xenograft mouse model. Whether long-acting E. coli-derived asparaginases will have similar anti-AML activity as a single agent or in combination with BCL-2 inhibition, and the potential of asparaginases to synergize with inhibitors of MCL1, a key resistance factor of BCL2 inhibition, has yet to be determined. Methods: Using a panel of 4 human AML cell lines (MOLM14, MV411, MonoMac6, HL60) as well as primary AML patient samples, weestablished the anti-leukemic activity of the BCL2 inhibitor S55746 and the MCL1 inhibitor S63845 alone and in combination with the long-acting E. coli asparaginase, calaspargase pegol-mknl (CalPegA). To determine the ability of S55746 or S63845 with or without CalPegA to inhibit mRNA translation, an established mechanism of Ven-PegC anti-leukemic activity, we measured the dissociation of the translation initiation factor eIF4E from 4EBP1, a negative regulator of cap-dependent mRNA translation, which then allows the formation of translation initiation complexes. The in vivo anti-leukemic efficacy of S55746 and S63845 alone and in combination with CalPegA was tested using an orthotopic AML xenograft mouse model with luciferase-expressing MV411 cells. Results: We found that CalPegA treatment resulted in a dose-dependent inhibition of AML cell proliferation, with IC 50 values ranging from 0.78 to 2.8 IU/mL. Single agent S55746 and S63845 treatment inhibited AML cell proliferation with IC 50 values ranging from 0.01 to 6.7 µM and 0.0001 to 0.08 µM, respectively. In 3 of the human AML cell lines (MOLM-14, MV4-11, and MonoMac6), the addition of a small dose (IC 20) of CalPegA potentiated the anti-leukemic effect of S55746, reducing the IC 50 by approximately 1- 4-fold, and by 10-fold in HL60 cells (Figure 1). This potentiation was also observed in 3 out of 4 primary AML patient samples tested, with the IC 50 of S55746 reduced by 4.5, 18, and 156-fold by the addition of CalPegA. The activity of S63845 was not substantially impacted by the addition of CalPegA in either AML cell lines (Figure 1) or primary samples. Immunoprecipitation of 4EBP1 revealed that treatment with a combination of either S63845 or S55746 with CalPegA increased the binding of eIF4E to 4EBP1 compared to single agent treatments, suggesting an inhibition of translational complex formation. In a MV411 tumor-bearing model, treatment withS55746-CalPegA combination reduced tumor burden over time significantly more than either single agent. S63845 treatment was highly effective at inhibiting leukemia growth over time and was not improved by the addition of CalPegA, as tumor burden in the S63845 and S63845 + CalPegA-treated groups was not significantly different over the course of the study (Figure 2). Conclusions: We report that the E. coli asparaginase, CalPegA, enhances the anti-leukemic effect of the BCL2 inhibitor, S55746, but does not impact the activity of the MCL1 inhibitor, S63845, in AML cell lines, patient derived primary AML samples, and in an AML xenograft mouse model. Ongoing studies are further investigating the anti-leukemic mechanism of S55476/S63845 + CalPegA and examining the efficacy and pharmacodynamic/pharmacokinetic effects of these agents in a patient derived xenograft model of AML.

Application of State Transition Theory and Treatment Modeling for Predicting Response to Chemotherapy in a Mouse Model of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, and the cancer originates in the bone marrow (BM) and circulates the body through the peripheral blood (PB), which can rapidly lead to BM failure and death. AML initiation and progression are composed of multiple biological properties, and we have previously shown that the state transition theory, state-space, and a model of the stochastic differential equation (SDE) could predict AML disease evolution using the mRNA of a conditional Cbfb-MYH11 (CM) knock-in mouse model. The model of the SDE represents AML evolution from health to disease as a state transition of the transcriptome state described as a particle undergoing Brownian motion in a double-well quasi-potential, where critical points define states of healthy state, c1, transition to AML, c2, and overt AML, c3. Along with the disease evolution of AML, prediction, and understanding of AML disease responses from chemotherapy aiming to provide optimized therapeutic strategies are required. Analyzing disease responses to chemotherapy from the state transition theory perspective will give us an understanding of post-chemotherapy disease dynamics and useful predictions which have the possibility to optimize treatment processes. To test the applicability of the AML state transition model, a time-series experiment using the conditional CM knock-in mouse model and state-transition modeling were performed. The mouse model was designed to recapitulate the human inv(16) AML, the leukemogenic fusion gene Cbfb-MYH11 (CM) in conditional CM knock-in mice (Cbfb56M/+/Mx1-Cre) cause the development of AML with a median survival of approximately 4 months after induction of CM. To induce CM expression, 6-8 weeks old CM knock-in mice were injected intraperitoneally with polyinosinic-polycytidylic acid [poly (I:C)] (InvivoGen, tlrl-picw-250) at 14 mg/kg/dose every other day for a total of 7 doses. CM leukemic mice were treated with a combination of cytarabine (50mg/kg/day; 5 days) and daunorubicin (1.5mg/kg/day; 3 days) after detection of overt leukemia, which is monitored by circulating leukemia blast (cKit+ > 20%) to model the 7+3 standard of care treatment for newly diagnosed AML. We used three independent experiments of mice as training and test cohorts (Group 1, Group 2, and Group 3). We collected 42 PBMC and 1 BM from CM-induced mice from Group 1 (4 AML and 1 control), 68 PBMC and 2 BM from Group 2 (4 AML), and 64 PBMC from Group 3 (3 AML and 2 control). The samples were collected weekly before, during, and following chemotherapy and subjected to RNA sequencing. The effects of chemotherapy were observed in the AML state-space with chronological time points as the transcriptome particle moving from leukemia state, c3, towards a state of healthy state, c1, crossing the unstable state, c2. The relapse was defined as the time of the first observation after the particle crosses back over unstable state, c2, towards the leukemic state, c3. The 10 AML mice out of 11 AML mice achieved a partial response with a mean time to relapse of 5 weeks. All 11 AML mice showed the anti-correlation between AML state in the state space was anti-correlated with the blast percentages (ckit%). We modified the state transition treatment model by introducing a parameter, γ, and the half-life of cytarabine (31.1 hours) and daunorubicin (21.9 hours). The gamma parameter is a force that works against the double-well quasi potential (γ >1) and represents the anti leukemogenic effects of cytarabine and daunorubicin. Gamma was determined by fitting the model to 6 AML mice data from the training cohort. The value was determined to be γ=2.7, providing a novel quantification of the treatment effect. We performed a mean arrival time analysis using simulations of the state transition treatment model, which accurately predicted the time to relapse with a prediction error of 6.6 days for the 3 AML mice from the test cohort. We could apply the state transition theory and propose the state transition treatment model to simulate the treatment process and the response from chemotherapy. Our model accurately predicted the time to respond to chemotherapy, from leukemia to health, and relapse, from health to leukemia, in the CM mouse model after chemotherapy. The state transition treatment model has implications for improving therapeutic strategies by targeting transcriptome state transition critical points in human AML.