Therapeutic Target In Hematologic Malignancies Research Articles

Alternative Splicing: A Potential Therapeutic Target in Hematological Malignancies

Leukemia represents the most prevalent malignancy in children, constituting 30% of childhood cancer cases, with acute lymphoblastic leukemia (ALL) being particularly heterogeneous. This paper explores the role of alternative splicing in leukemia, highlighting its significance in cancer development and progression. Aberrant splicing is often driven by mutations in splicing-factor genes, which can lead to the production of variant proteins that contribute to oncogenesis. The spliceosome, a complex of small nuclear RNAs and proteins, facilitates RNA splicing, a process critical for generating diverse mRNA and protein products from single genes. Mutations in splicing factors, such as U2AF1, SF3B1, SRSF2, ZRSR2, and HNRNPH1, are frequently observed across various hematological malignancies and are associated with poor prognosis and treatment resistance. This research underscores the necessity of understanding the mechanisms of RNA splicing dysregulation in order to develop targeted therapies to correct these aberrant processes, thereby improving outcomes for patients with leukemia and related disorders.

Advances and clinical applications of immune checkpoint inhibitors in hematological malignancies.

Immune checkpoints are differentially expressed on various immune cells to regulate immune responses in tumor microenvironment. Tumor cells can activate the immune checkpoint pathway to establish an immunosuppressive tumor microenvironment and inhibit the anti-tumor immune response, which may lead to tumor progression by evading immune surveillance. Interrupting co-inhibitory signaling pathways with immune checkpoint inhibitors (ICIs) could reinvigorate the anti-tumor immune response and promote immune-mediated eradication of tumor cells. As a milestone in tumor treatment, ICIs have been firstly used in solid tumors and subsequently expanded to hematological malignancies, which are in their infancy. Currently, immune checkpoints have been investigated as promising biomarkers and therapeutic targets in hematological malignancies, and novel immune checkpoints, such as signal regulatory protein α (SIRPα) and tumor necrosis factor-alpha-inducible protein 8-like 2 (TIPE2), are constantly being discovered. Numerous ICIs have received clinical approval for clinical application in the treatment of hematological malignancies, especially when used in combination with other strategies, including oncolytic viruses (OVs), neoantigen vaccines, bispecific antibodies (bsAb), bio-nanomaterials, tumor vaccines, and cytokine-induced killer (CIK) cells. Moreover, the proportion of individuals with hematological malignancies benefiting from ICIs remains lower than expected due to multiple mechanisms of drug resistance and immune-related adverse events (irAEs). Close monitoring and appropriate intervention are needed to mitigate irAEs while using ICIs. This review provided a comprehensive overview of immune checkpoints on different immune cells, the latest advances of ICIs and highlighted the clinical applications of immune checkpoints in hematological malignancies, including biomarkers, targets, combination of ICIs with other therapies, mechanisms of resistance to ICIs, and irAEs, which can provide novel insight into the future exploration of ICIs in tumor treatment.

Advancements in Personalized CAR-T Therapy: Comprehensive Overview of Biomarkers and Therapeutic Targets in Hematological Malignancies.

Chimeric antigen receptor T-cell (CAR-T) therapy is a novel anticancer therapy using autologous or allogeneic T-cells. To date, six CAR-T therapies for specific B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma (MM) have been approved by the Food and Drug Administration (FDA). Significant barriers to the effectiveness of CAR-T therapy include cytokine release syndrome (CRS), neurotoxicity in the case of Allogeneic Stem Cell Transplantation (Allo-SCT) graft-versus-host-disease (GVHD), antigen escape, modest antitumor activity, restricted trafficking, limited persistence, the immunosuppressive microenvironment, and senescence and exhaustion of CAR-Ts. Furthermore, cancer drug resistance remains a major problem in clinical practice. CAR-T therapy, in combination with checkpoint blockades and bispecific T-cell engagers (BiTEs) or other drugs, appears to be an appealing anticancer strategy. Many of these agents have shown impressive results, combining efficacy with tolerability. Biomarkers like extracellular vesicles (EVs), cell-free DNA (cfDNA), circulating tumor (ctDNA) and miRNAs may play an important role in toxicity, relapse assessment, and efficacy prediction, and can be implicated in clinical applications of CAR-T therapy and in establishing safe and efficacious personalized medicine. However, further research is required to fully comprehend the particular side effects of immunomodulation, to ascertain the best order and combination of this medication with conventional chemotherapy and targeted therapies, and to find reliable predictive biomarkers.

RPRD1B/CREPT facilitates the progression of diffuse large B-cell lymphoma by inhibiting apoptosis through the NF-κB signaling pathway

Objective: To investigate the role of RPRD1B in the progression of diffuse large B-cell lymphoma (DLBCL) and its potential as a therapeutic target. Methods: This study analyzed RPRD1B expression in DLBCL and normal tissues using public databases and assessed its prognostic impact through survival analysis. In vitro and in vivo experiments were conducted to explore the mechanisms by which RPRD1B influences tumor growth and apoptosis. Results: RPRD1B expression was significantly elevated in DLBCL compared to normal tissues and was associated with poor prognosis. In vitro and in vivo experiments demonstrated that RPRD1B promoted lymphoma cell proliferation and inhibited apoptosis through the NF-κB signaling pathway. Conclusions: RPRD1B plays a critical role in the progression of DLBCL by modulating apoptosis and cellular proliferation. Targeting RPRD1B may offer a novel therapeutic strategy for DLBCL, suggesting its potential as a prognostic marker and therapeutic target in hematological malignancies.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

Discovery of First-in-Class Small Molecule Inhibitors of the IRF4-PU.1/Spi-B Interaction

Introduction Many transcription factors (TF) are promising therapeutic targets in hematological malignancies but have been mislabeled as ‘undruggable’ due to their dynamic structures and lack of feasible paths for bioactive small molecules. Multiple myeloma (MM), the second most common hematological malignancy, is predominantly driven by oncogenic transcription factors such as c-Myc & interferon regulatory factor 4 (IRF4). Attempts at targeting IRF4 have relied on immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, which target upstream TFs of IRF4 - IKZF1 & IKZF3. Unfortunately, IMiDs are highly susceptible to resistance mechanisms in the clinic and fail to provide deep and sustained downregulation of IRF4 due to theirindirect mechanism of action. Currently, no known small molecules that directly bind and inhibit IRF4 exist. Moreover, no probes or screening based assays have been developed for IRF4 or any member of the IRF family.IRF4 is the top ranked dependency of MM and is a validated clinical target given the success of IMiD-based regimens. Thus, orally available small molecules that can directly target IRF4 will have significant benefit for patients with MM. Methods & Results To identify small molecules that inhibit or bind IRF4, we first cloned, expressed, and purified numerous constructs of the interferon accessory domain (IAD - residues 238-420) and full-length IRF4 (residues 1-451). To enable traditional high-throughput screening (HTS) campaigns, we first identified peptide ligands of IRF4 based on the IRF4 - PU.1/Spi-B interaction, an oncogenic signaling node in various hematological malignancies. We profiled peptides derived from PU.1 and Spi-B and confirmed binding to the IAD domain with K ds of 1 - 7 µM by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). Encouraged by the robust signal seen with our FP assay, we further developed a TR-FRET based method using a CoraFluor labeled IAD domain which significantly boosted our signal-to-noise ratio for our HTS efforts (Z' > 0.8). We screened this interaction against the Chembridge2020 drug library, which consists of 50,000 lead-like small molecules. The screen afforded 68 small molecules that had inhibition > 50% by TR-FRET (hit rate of ~0.1%). We prioritized our most potent compound, HIT-1, which has a TR-FRET IC 50 of 2 µM. Further binding validation including ITC, biotin-based pull-downs in human myeloma cell lines (HMCLs), and a BRET based target engagement assay confirm binding to both recombinant and full-length IRF4. Additionally, medicinal chemistry optimization led to the development of 1, with sub-micromolar affinity to IRF4, and an inactive control 2. Biological assessment of compounds 1 and inactive control 2 are underway in HMCLs; initial results show on-target modulation of IRF4 signaling processes. Compound 1 represents the first validated small molecule targeting the ‘undruggable’ IRF family and warrants its use in investigating the IRF4-PU.1/Spi-B interaction in oncology and immunology. Conclusions These small molecules are the first inhibitors of the IRF4-PU.1 interaction. We have developed novel biochemical assays that can be applied to other IRF family members, including IRF8. Additionally, we showcase translatable strategies to build novel biochemical assays for other transcription factors, leveraging known PPIs for probe development. These ligands represent starting points for hetero-bifunctional compounds and chemical probes for modulating IRF4 activity both in vitro and in vivo.

Targeting integrated stress response with ISRIB combined with imatinib treatment attenuates RAS/RAF/MAPK and STAT5 signaling and eradicates chronic myeloid leukemia cells

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however, the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously, we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover, the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here, we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways, which are recognized as drivers of resistance. Mechanistically, this drug combination attenuated both interacting signaling networks, leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently, leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK, JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation, viability and the stress response. Collectively, these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment.

Protein Arginine Methyltransferases as Therapeutic Targets in Hematological Malignancies.

Arginine methylation is a common post-translational modification affecting protein activity and the transcription of target genes when methylation occurs on histone tails. There are nine protein arginine methyltransferases (PRMTs) in mammals, divided into subgroups depending on the methylation they form on a molecule of arginine. During the formation and maturation of the different types of blood cells, PRMTs play a central role by controlling cell differentiation at the transcriptional level. PRMT enzymatic activity is necessary for many cellular processes in hematological malignancies, such as the activation of cell cycle and proliferation, inhibition of apoptosis, DNA repair processes, RNA splicing, and transcription by methylating histone tails' arginine. Chemical tools have been developed to inhibit the activity of PRMTs and have been tested in several models of hematological malignancies, including primary samples from patients, xenografts into immunodeficient mice, mouse models, and human cell lines. They show a significant effect by reducing cell viability and increasing the overall survival of mice. PRMT5 inhibitors have a strong therapeutic potential, as phase I clinical trials in hematological malignancies that use these molecules show promising results, thus, underlining PRMT inhibitors as useful therapeutic tools for cancer treatment in the future.

Role of reactive oxygen species in regulating 27-hydroxycholesterol-induced apoptosis of hematopoietic progenitor cells and myeloid cell lines

Oxysterols are oxygenated derivatives of cholesterol that contain an additional hydroxy, epoxide, or ketone group in the sterol nucleus and/or a hydroxyl group in the side chain of the cholesterol molecule. 27-Hydroxycholesterol (27HC) is a side-chain oxysterol that is oxygenated at the 27th carbon atom of cholesterol. The oxysterol (27HC) is produced via oxidation by sterol 27-hydroxylase (CYP27A1) and metabolized via oxysterol 7a-hydroxylase (CYP7B1) for bile acid synthesis in the liver. A previous study has demonstrated that treatment with the alternative Estrogen receptor alpha (ERα) ligand 27HC induces ERα-dependent hematopoietic stem cell (HSC) mobilization. In addition, Cyp27a1-deficient mice demonstrate significantly reduced 27HC levels and HSC mobilization. Here, we report that exogenous 27HC treatment leads to a substantial reduction in the hematopoietic stem and progenitor cell (HSPC) population owing to significantly increased reactive oxygen species (ROS) levels and apoptosis in the bone marrow (BM). However, 27HC does not influence the population of mature hematopoietic cells in the BM. Furthermore, exogenous 27HC treatment suppresses cell growth and promotes ROS production and apoptosis in leukemic cells. Moreover, acute myeloid leukemia (AML) patients with high CYP7B1 expression (expected to have inhibition of 27HC) had significantly shorter survival than those with low CYP7B1 expression (expected to have an elevation of 27HC). Single-cell RNA-sequencing (scRNA seq) analysis revealed that the expression of CYP7B1 was significantly increased in AML patients. Thus, our study suggests that 27HC may serve as a potent agent for regulating pools of HSPCs and may have an application as a novel therapeutic target for hematological malignancies. Collectively, pharmacological inhibition of CYP7B1 (expected to have an elevation of 27HC) would potentially have fewer long-term hematological side effects, particularly when used in combination with chemotherapy or radiation for the treatment of leukemia patients.

Autophagy in Hematological Malignancies.

Simple SummaryAutophagy is a dynamic and tightly regulated process that seems to have dual effects in cancer. In some contexts, it can induce carcinogenesis and promote cancer cell survival, whereas in others, it acts preventing tumor cell growth and tumor progression. Thus, autophagy functions seem to strictly depend on cancer ontogenesis, progression, and type. Here, we will dive into the current knowledge of autophagy in hematological malignancies and will highlight the main genetic components involved in each cancer type.Autophagy is a highly conserved metabolic pathway via which unwanted intracellular materials, such as unfolded proteins or damaged organelles, are digested. It is activated in response to conditions of oxidative stress or starvation, and is essential for the maintenance of cellular homeostasis and other vital functions, such as differentiation, cell death, and the cell cycle. Therefore, autophagy plays an important role in the initiation and progression of tumors, including hematological malignancies, where damaged autophagy during hematopoiesis can cause malignant transformation and increase cell proliferation. Over the last decade, the importance of autophagy in response to standard pharmacological treatment of hematological tumors has been observed, revealing completely opposite roles depending on the tumor type and stage. Thus, autophagy can promote tumor survival by attenuating the cellular damage caused by drugs and/or stabilizing oncogenic proteins, but can also have an antitumoral effect due to autophagic cell death. Therefore, autophagy-based strategies must depend on the context to create specific and safe combination therapies that could contribute to improved clinical outcomes. In this review, we describe the process of autophagy and its role on hematopoiesis, and we highlight recent research investigating its role as a potential therapeutic target in hematological malignancies. The findings suggest that genetic variants within autophagy-related genes modulate the risk of developing hemopathies, as well as patient survival.

The hedgehog pathway in hematopoiesis and hematological malignancy.

The Hedgehog (HH) pathway is a promising therapeutic target in hematological malignancies. Activation of the pathway has been tied to greater chances of relapse and poorer outcomes in several hematological malignancies and inhibiting the pathway has improved outcomes in several clinical trials. One inhibitor targeting the pathway via the protein Smoothened (SMO), glasdegib, has been approved by the FDA for use with a low dose cytarabine regiment in some high-risk acute myeloid leukemia patients (AML). If further clinical trials in glasdegib produce positive results, there may soon be more general use of HH inhibitors in the treatment of hematological malignancies.While there is clinical evidence that HH inhibitors may improve outcomes and help prevent relapse, a full understanding of any mechanism of action remains elusive. The bulk of AML cells exhibit primary resistance to SMO inhibition (SMOi), leading some to hypothesize that that clinical activity of SMOi is mediated through modulation of self-renewal and chemoresistance in rare cancer stem cells (CSC). Direct evidence that CSC are being targeted in patients by SMOi has proven difficult to produce, and here we present data to support the alternative hypothesis that suggests the clinical benefit observed with SMOi is being mediated through stromal cells in the tumor microenvironment.This paper’s aims are to review the history of the HH pathway in hematopoiesis and hematological malignancy, to highlight the pre-clinical and clinical evidence for its use a therapeutic target, and to explore the evidence for stromal activation of the pathway acting to protect CSCs and enable self-renewal of AML and other diseases. Finally, we highlight gaps in the current data and present hypotheses for new research directions.

Targeting PIM2 by JP11646 results in significant antitumor effects in solid tumors.

Proviral integration of Moloney virus 2 (PIM2) is a pro-survival factor of cancer cells and a possible therapeutic target in hematological malignancies. However, the attempts at inhibiting PIM2 have yielded underwhelming results in early clinical trials on hematological malignancies. Recently, a novel pan-PIM inhibitor, JP11646, was developed. The present study examined the utility of targeting PIM2 in multiple solid cancers and investigated the antitumor efficacy and the mechanisms of action of JP11646. When PIM2 expression was compared between normal and cancer tissues in publicly available datasets, PIM2 was found to be overexpressed in several types of solid cancers. PIM2 ectopic overexpression promoted tumor growth in in vivo xenograft breast cancer mouse models. The pan-PIM inhibitor, JP11646, suppressed in vitro cancer cell proliferation in a concentration-dependent manner in multiple types of cancers; a similar result was observed with siRNA-mediated PIM2 knockdown, as well as an increased in cell apoptosis. By contrast, another pan-PIM inhibitor, AZD1208, suppressed the expression of downstream PIM2 targets, but not PIM2 protein expression, corresponding to no apoptosis induction. As a mechanism of PIM2 protein degradation, it was found that the proteasome inhibitor, bortezomib, reversed the apoptosis induced by JP11646, suggesting that PIM2 degradation by JP11646 is proteasome-dependent. JP11646 exhibited significant anticancer efficacy with minimal toxicities at the examined doses and schedules in multiple in vivo mice xenograft solid cancer models. On the whole, the present study demonstrates that PIM2 promotes cancer progression in solid tumors. JP11646 induces apoptosis at least partly by PIM2 protein degradation and suppresses cancer cell proliferation in vitro and in vivo. JP11646 may thus be a possible treatment strategy for multiple types of solid cancers.

Abstract 6395: Butyrophilins 3A as an immune checkpoint and a therapeutic target in hematological malignancies

Abstract Introduction: Butyrophilins (BTN) are a family of immune regulators in the immunoglobin superfamily, structurally similar to the B7 family. BTN3A, highly expressed in a variety of human cancer cells, suppresses αβ T cell activation by preventing CD45 segregation and immune synapse formation. Human BTN3A has three isoforms with almost identical extracellular domains, but each has unique cytoplasmic tail. BTN3A is highly expressed in a variety of human cancer cells. Therefore, we hypothesize that blocking BTN3A can remove the immunosuppressive effects of cancer cells and therefore enhance the tumor killing effects of immune cells. Methods: The expression of BTN3A on primary human immune cells was examined by flow cytometry. Human BTN3A2-IgG Fc fusion protein was generated in the Drosophila expression system. BALB/c wild type mice were immunized with the fusion protein and hybridomas were generated. High-affinity monoclonal antibodies (mAb) were screened and purified. NIH 3T3 cells transfected with human BTN3A1, BTN3A2 or BTN3A3 were used to examine if the purified mAbs can recognize each isoform on cell surface. Artificial CHO antigen-presenting cells (APC CHO cells) were transfected with or without hBTN3A2, and then co-cultured with human primary T cells in the presence of mouse IgG isotype or anti-hBTN3A2 mAb. A mixed lymphocyte reaction with different donor dendritic cells and T cells was performed with isotype or anti-hBTN3A mAb. The hBTN3A2 mRNA expression in a variety of tumor cell lines was screened in a gene database, and the protein expression of selected tumor cell lines was examined by flow cytometry with anti-hBTN3A mAb. Findings: Multiple tumor cell lines, originating from hematologic malignancies, carcinomas, or sarcomas, showed high cell surface expression of hBTN3A by flow cytometry. Two clones of high-affinity, anti-hBTN3A mAb were generated and purified. Both are IgG1 isotypes. Anti-hBTN3A mAb bound to BTN3A1, BTN3A2 or BTN3A3 expressed on NIH 3T3 cells. The majority of human immune cells isolated from peripheral blood from healthy donors expressed hBTN3A, including CD4 and CD8 T cells, B cells, monocytes, natural killer cells and γδ T cells. BTN3A2 expressing APC CHO cells inhibited primary αβ T cell activation, and anti-BTN3A mAb restored T cell proliferation as well as stimulated cytotoxic cytokine production in some healthy donors. Conclusion: BTN3A is an immunosuppressive receptor that inhibits αβ T cell activation. Anti-hBTN3A mAb restores T cell activation, suggesting the potential for BTN3A to be a target for cancer immunotherapy. Citation Format: Yixian Li, Xiaoxin Ren, Yao Wei, Mou Peng, Hao Wang, Marc Christopher Pulanco, Christopher David Nishimura, Philip Galbo, Anne Madsen, R. Alejandro Sica, Xingxing Zang. Butyrophilins 3A as an immune checkpoint and a therapeutic target in hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6395.

Abstract 823: ABL1 kinase regulates leukemia phenotype and response to therapy

Abstract The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase. Cytoplasmic ABL1 stimulates, whereas nuclear ABL1 inhibits cell growth. Analysis of the CGAP Mitelman database detected loss of ABL1 allele in hematologic malignancies. Among the top genetic aberrations detected in acute myeloid leukemias were t(8;21) generating fusion protein AML1-ETO and NUP98 translocations, e.g., t(1;11) encoding NUP98-PMX1.Expression of AML1-ETO and NUP98-PMX1 in Lin-cKit+ stem cell enriched bone marrow cells from Abl1-/-;Vav-Cre and Abl1+/+;Vav-Cre mice revealed that oncogene-expressing Abl1-/- cells gained tremendous proliferation advantage. Conversely, overexpression of ABL1 and DPH (ABL1 kinase agonist)-mediated activation of ABL1 kinase resulted in reduced proliferation of AML1-ETO and NUP98-PMX1 cells. Since the role of ABL1 in DNA damage response (DDR) is well established, we applied the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi) to identify potential therapeutic vulnerabilities. AML1-ETO;Abl1-/- cells were more sensitive to all three inhibitors when compared to AML1-ETO;Abl1+/+ cells. Remarkably, NUP98-PMX1;Abl1-/- cells were exceptionally sensitive to ATRi, but not to ATMi and DNA-PKi when compared to NUP98-PMX1;Abl+/+ cells. ABL1 did not affect the sensitivity of transformed cells to DNA damaging agent doxorubicin. To pinpoint mechanisms collaborating with Abl1 deletion in transformation of hematopoietic cells, Abl1 was knockout in 32Dcl3 cells by CRISPR/Cas9 to obtain 32Dcl3-Abl1ko cells. 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells were challenged to induce transformation by starving them from growth factors (IL3). Only 32Dcl3-Abl1ko cell populations generated growth factor-independent cells. This indicates that the absence of Abl1 may facilitate malignant transformation of hematopoietic cells. RNA-seq was performed to compare gene expression in 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells in the presence or absence of IL-3. Pathway analysis of FPKM reads (top 30 significant canonical pathways) and Venn diagram analysis followed by KEGG pathway analysis (291 genes uniquely expressed in growth factor-independent 32Dcl3-Abl1ko cells) implicated PI3K-AKT pathway signaling including mTOR and sirtuins (SITR) in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib, mTOR inhibitor rapamycin and SIRT2 inhibitor thiomyristoyl exerted selective activity against Lin-cKit+ AML1-ETO;Abl1-/- and/or NUP98-PMX1;Abl1-/- cells when compared to Abl1+/+ counterparts. Moreover, inhibition of ABL1 kinase by imatinib in Lin-cKit+ AML1-ETO;Abl1+/+ and/or NUP98-PMX1;Abl+/+ cells induced the sensitivity to PI3K-ATK pathway inhibitors. In conclusion, we showed that normal ABL1 kinase plays a tumor suppressor role and is a potential therapeutic target in hematological malignancies induced by AML1-ETO and NUP98-PMX1. The latter statement is being currently tested in animal models. Citation Format: Konstantin Golovine, Zhaorui Lian, Kumaraswamy Naidu Chitrala, Gleb Ablakov, Margaret Nieborowska-Skorska, Jian Huang, Tomasz Skorski. ABL1 kinase regulates leukemia phenotype and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 823.

Exportin 1 as a therapeutic target against chemoresistance in small cell lung cancer.

e20613 Background: Small cell lung cancer (SCLC) is an extremely aggressive disease comprising around 13% of lung cancer cases and responsible for approximately 250,000 deaths globally per year. A major cause of SCLC poor prognosis is the sparse treatment options available, typically resulting in only transient responses. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Methods: We performed an in vitro CRISPR screen in SCLC cell lines to identify potential therapeutic targets to sensitize to chemotherapy, including short-term cultured cell lines derived from patient-derived xenografts (PDXs). Candidate hits were validated genetically and pharmacologically with in vitro and in vivo. Signaling pathways involved in phenotypes observed were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Results: Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. Importantly, exportin 1 is a therapeutic target in hematological malignancies, counting with a potent and selective inhibitor, selinexor, approved for clinical use. Combination of selinexor with cisplatin or irinotecan demonstrated high synergy in vitro and exquisite efficacy i n vivo in an array of chemonäive and chemoresistant SCLC PDXs, respectively, including all major SCLC subtypes. These chemotherapy-sensitizing effects were associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation, potentially occurring as a cytoprotective/anti-apoptotic mechanism in response to chemotherapy. The combinations were well tolerated in mice. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor type or hematological malignancies, which we were able to confirm at the protein level in clinical TMAs. Conclusions: Exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively. These results provide preclinical rationale for the combination of selinexor with first line and second line chemotherapy in SCLC. The clinical availability of selinexor will allow immediate clinical translation of these results in a disease setting with extremely limited therapeutic options.

Discovery of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as potent and selective CDK9 inhibitors that enable transient target engagement for the treatment of hematologic malignancies

Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and a potential therapeutic target in hematologic malignancies. Selective and transient CDK9 inhibition reduces Mcl-1 expression and induces apoptosis in Mcl-1-dependent tumor cells for survival. Here, we describe our efforts to discover a novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one as CDK9 inhibitors. Compound 32k was identified as a selective CDK9 inhibitor with short pharmacokinetic and physicochemical properties suitable for intravenous administration. Short-term treatment with 32k resulted in a rapid dose-dependent decrease in cellular p-Ser2-RNAPII, Mcl-1 and c-Myc, leading to apoptosis in the MV4-11 cell line. Correspondingly, significant in vivo antitumor efficacy was observed in xenograft models derived from multiple hematological tumors with intermittent 32k dosing. These results provide evidence that selective transient CDK9 inhibitors could be used for the treatment of hematologic malignancies.

Development of Anti-CD99scFvs for the Treatment of Acute Myeloid Leukemia

CD99 has gained much attention in recent years as a novel therapeutic target in hematological malignancies, due to its upregulation in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). We have recently shown that targeting CD99 with a knockdown approach or with commercial antibodies results in antileukemia activity in AML cells. We have also developed anti-CD99 nanoparticles and demonstrated excellent in vitro and in vivo antileukemia activity. Here we report the development of human single chain variable fragment targeting CD99 (anti-CD99 scFv) and the preclinical activity in AML cells and in AML xenograft mouse model. The anti-CD99 scFv was developed by inserting CD99scFv DNA sequence into the pFUSE vector, encoding pATL103-CD99 scFvs. To produce anti-CD99 scFv, the plasmids were transfected in Expi293 (HEK293T) cells. Then the medium was harvested and underwent two cycles of dialysis. To determine product purity, proteins were analyzed on SDS-PAGE gel stained with Coomassie blue. For each 240ml cell culture media with recombinant CD99 plasmids, we obtained about 1-2mg anti-CD99 scFv. The binding affinity of CD99 scFv to CD99 surface protein was assessed in 293T cells (CD99 null cells) which exhibited no binding and in MOLM-13 cells and MV4-11 (CD99 positive AML cells) which demonstrated strong binding. Treatment with 5uM of anti-CD99 scFv significantly reduced cell viability in both leukemic cell line MOLM-13 and MV4-11 (MOLM-13: 35.14%, P= 0.008; MV4-11: 30.17%, P=0.002) and primary AML patient blasts (29.37%, P=0.048) compared with control cells. Colony forming assay showed that anti-CD99 scFv treated AML blasts exhibited less number of colonies compared with control cells (plating efficiency (PE): 0.035% vs 0.12%). We also established the in vivo antileukemia activity of anti-CD99 scFv using MOLM-13 cells (FLT3-ITD positive AML cells) NOD scid gamma (NSG) xenograft mouse model. MOLM-13 (2.5x10^6) cells were engrafted into NSG mice via IV tail injection (N=4 mice per group). Mice were treated with PBS (group 1) or 4mg/kg of anti-CD99 scFV on days 10, 14, 18 and 22 post cell engraftment. Mice were euthanized on day 24 and levels of leukemia engraftment were assessed by flow cytometry measurements of huCD45 staining of cells collected from the bone marrow and the peripheral blood and compared between the two groups. Mice treated with four doses of 4mg/kg CD99scFv demonstrated significant reduction in leukemia engraftment in the bone marrow assessed by flow cytometry measurements of huCD45 staining compared with the PBS mice group (huCD45%: 39.7 vs 56, P = 0.0017). In conclusion, we report the development of anti-CD99 single chain variable fragments for the treatment of AML. Our study demonstrates good binding affinity and specificity and a promising preclinical antileukemia activity both in AML cells and in xenograft mouse model. DisclosuresYaghmour: Jazz: Consultancy, Honoraria; Astellas: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

P21-Activated kinases as promising therapeutic targets in hematological malignancies.

The p21-Activated Kinases (PAKs) are a family of six serine/threonine kinases that were originally identified as downstream effectors of the Rho GTPases Cdc42 and Rac. Since the first PAK was discovered in 1994, studies have revealed their fundamental and biological importance in the development of physiological systems. Within the cell, PAKs also play significant roles in regulating essential cellular processes such as cytoskeletal dynamics, gene expression, cell survival, and cell cycle progression. These processes are often deregulated in numerous cancers when different PAKs are overexpressed or amplified at the chromosomal level. Furthermore, PAKs modulate multiple oncogenic signaling pathways which facilitate apoptosis escape, uncontrolled proliferation, and drug resistance. There is growing insight into the critical roles of PAKs in regulating steady-state hematopoiesis, including the properties of hematopoietic stem cells (HSC), and the initiation and progression of hematological malignancies. This review will focus on the most recent studies that provide experimental evidence showing how specific PAKs regulate the properties of leukemic stem cells (LSCs) and drug-resistant cells to initiate and maintain hematological malignancies. The current understanding of the molecular and cellular mechanisms by which the PAKs operate in specific human leukemia or lymphomas will be discussed. From a translational point of view, PAKs have been suggested to be critical therapeutic targets and potential prognosis markers; thus, this review will also discuss current therapeutic strategies against hematological malignancies using existing small-molecule PAK inhibitors, as well as promising combination treatments, to sensitize drug-resistant cells to conventional therapies. The challenges of toxicity and non-specific targeting associated with some PAK inhibitors, as well as how future approaches for PAK inhibition to overcome these limitations, will also be addressed.

Abstract 385: Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma

Abstract Background: IRAK4, a serine/threonine kinase, was identified as a therapeutic target in hematological malignancies. IRAK4-mediated activation of NF-kappaB could play an important role in NF-kappaB-regulated survival and chemoresistance of cancer cells. CA-4948, a first-in-class small molecule inhibitor of IRAK4, has been tested in Phase 1 clinical trial with evidence of clinical activity in Non-Hodgkin's Lymphoma (NHL) patients. To support the development of companion diagnostic for CA-4948, we developed an immunohistochemical (IHC) assay and explored expression of potential biomarkers of response to CA-4948 in tumor biopsy samples obtained from NHL patients. Materials and Methods: IHC assay was developed and used for analysis of NF-kappaB p-p50 and p-IRAK1 expression in tumor biopsy samples obtained from 14 patients with NHL. Patients were defined according to their clinical response to CA-4948 treatment: stable disease (SD), 6 cases and progressive disease (PD), 8 cases. Results: We found nuclear and/or cytoplasmic expression of NF-kappaB p-p50 in all 6 SD cases treated with 50 mg QD (2 cases, tumor regression), 50 mg BID (1 case), 200 mg BID (1 case, tumor regression) and 400 mg BID (2 cases). Expression of NF-kappaB p-p50 was not detected in 7 of 8 cases with PD including patients treated with 50 mg QD (1 case), 100 mg QD (1 case), 100 mg BID (3 cases), 200 mg BID (1 case) and 400 mg BID (1 case). We found statistically significant correlation between expression of NF-kappaB p-p50 in tumor biopsy and SD in NHL patients treated with CA-4948 (p<0.05). Analysis of NF-kappaB p-p50 expression in paired tumor biopsy samples (3 cases) collected before and after the treatment with CA-4948 revealed a significant downregulation of NF-kappaB p-p50 expression in tumors obtained from CA-4948-treated NHL patients. In support of our in vivo findings, our in vitro experiments demonstrated that expression of NF-kappaB p-p50 was depleted in 3D lymphoma organoids treated with a clinically relevant concentration of CA-4948. Our results support further development of NF-kappaB p-p50 as a potential predictive and pharmacodynamic biomarker of IRAK4 inhibitor CA-4948. We found that expression of p-IRAK1 in tumor biopsies was not correlated with clinical response. Conclusions: Although the cohort size is small, our findings suggest that expression of NF-kappaB p-p50 can serve as biomarker to predict SD in response to the treatment with IRAK4 inhibitor CA-4948 in NHL patients. NF-kappaB p-p50 selection strategy might be used in future clinical trials to identify NHL patients which are most likely to respond to CA-4948 in combination with chemotherapy or targeted therapeutics. Citation Format: Andrey Ugolkov, Maria Samson, Rosanna Hok, Reinhard von Roemeling, Robert Martell. Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 385.

Ruxolitinib in Combination with Vorinostat Suppresses Tumor Growth and Alters the Metabolic Phenotype in Haematological Diseases

Background and Objectives Jak-2 dysregulation has a significative role as an oncogenic driver and as a prominent therapeutic target in hematological malignancies. Ruxolitinib is pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2 and minor activity against JAK3. Vorinostat is an HDAC inhibitor able to reduce Jak-2 expression affecting Jak-2 mRNA expression and increasing Jak-2 proteasomal deterioration. In this study we hypothesized that Ruxolitinib and Vorinostat combination could have a synergistic effects on haematological disease. Methods. We tested Ruxolitinib and Vorinostat alone and in combination for 24 and 48 hrs in 12 lymphoma and myeloma cell lines. The synergism was assessed by Chou-Talalay method. The cell cycle, the apoptosis, the caspase activation, the autophagy, the interleukin expression, the ROS generation, the ATP levels, the lactate, the phosphorylation status of protein kinases were evaluated in all 12 cell lines after 24 hrs. Co-coltures with bone marrow stromal cells were also performed after 24 to 120 hrs of treatments. Results. Treatment of 12 cell lines with low doses of Ruxolitinib and Vorinostat as single agents for 24h was rather ineffective at blocking cell growth if compared with the efficacy of the combination of the two drugs. The combination was additive in two lines of mantle cell lymphoma, in one line of Hodgkin lymphoma, in one line of myeloma, in one line of T cell lymphoma and in on line of B cell lymphoma. In the remaining cell lines the combination of the two drugs was highly synergistic. This synergistic cytotoxic action was maintained over time up to 120 hrs also in the presence of stromal cells. Ruxolitinib and Vorinostat combination influenced the cell cycle distribution increasing G2-M phase arrest in all 12 cell lines after 24 hrs. The levels of p21 and p27 resulted up-regulated while the levels of Cyclin D and Aurora A were downregulated in all 12 cell line after combined treatment. The apoptotic AnnexinV-positive cells after 24 hrs of treatment with Ruxolitinib or Vorinostat alone was less than 10%. However, the percentage of apoptotic cells grew up to 50 % after treatment of the cells with Ruxolitinib in combination with Vorinostat. After 24 of combined treatment, we detected a cleavage of caspase 8 and caspase 3 in all 12 cell line, suggesting activation of the extrinsic apoptotic pathway. Quantification of caspase 9 in all 12 cell lines has resulted in observation of an increase in cleaved form only in the six most sensitive cell lines, suggesting a caspase activation mitochondrial events regulating the intrinsic apoptosis. Bax, Bid and BAD levels increased in all 12 cell lines treated with combined drugs, but apoptotic inhibitors BLC-2 and MCL-1 were down regulated only in the 6 more sensitive cell lines. Our experiment showed that Ruxolitinib in combination with Vorinostat reduced IL-10 and IL-6 secretion in all 12 cell lines in a comparable way. The expression of IL-17A after combined treatment was significantly decreased in only the 6 most sensitive cell lines. Ruxolitinib and Vorinostat alone and in combination induced a decrease of the amount of autophagy-related protein p62 in cancer cells, indicating the activation of autophagic system. Autophagy and apoptosis increased the oxidative stress leading to ROS production and in this work, we showed that Ruxolitinib and Vorinostat synergistically induce apoptosis in 6 more sensitive cell lines, associated with a marked increase in ROS generation. Interestingly, our data have shown that combined therapy alters tumor cell metabolism by reducing the glycolic state by causing greater sensitivity of the six cell lines to the combined treatment. Conclusions. In conclusions, the combination of Ruxolitinib and Vorinostat is able to decrease glucose metabolism and this partial reversion of Warburg effect is associated to ROS production, apoptosis and cell growth inhibition. The drugs tested under different cell culture conditions provide informations on drug efficacy and could help predict pharmacological effects that may be difficult to translate from in vitro and ex vivo conditions to the clinic. This study gives evidence of synergistic interaction between Ruxolitinib and Vorinostat in lymphoid cells and provides the rationale for clinical studies with the combination of both agents in patients. DisclosuresNo relevant conflicts of interest to declare.