Target In Chronic Lymphocytic Leukemia Research Articles

Abstract PO5-05-08: LOSS OF SINGLEMINDED 2S RESULTS IN A PI3K SUBUNIT SWITCH WHICH DRIVES THERAPEUTIC RESISTANCE IN ESTROGEN RECEPTOR POSITIVE BREAST CANCER

Abstract Estrogen receptor (ER) + breast cancer (BC) comprises over 70% of BC cases and are targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases are commonly diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastases. We have identified a tumor suppressor gene, singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits epithelial to mesenchymal transition and metastasis, and is downregulated in the progression of breast disease. In ER+BC cell lines, loss of SIM2s results in upregulation of mesenchymal markers and increased PI3K/Akt signaling. Dysregulation of the PI3K/Akt signaling pathway in ER+BC is involved with tumor progression and acquired therapeutic resistance. Our study suggests loss of SIM2s confers resistance in ER+BC through a PI3K subunit switch resulting in upregulation of pro-survival signaling. MCF7 SIM2 knock out cells exhibit increased expression of mesenchymal markers and undergo a phenotypic change compared to wild type cells. Utilizing a migration/invasion assay, MCF7 SIM2 knock out cells exemplify an increase in invasion potential compared to wild type cells. Furthermore, changes in PI3K subunit expression were observed via western blot and real time qPCR analysis. Clonogenic assays revealed an acquired resistance to PI3Kα inhibition, but a susceptibility to PI3Kδ inhibition. This is a significant finding as a current standard of care for patients with ER+ breast cancer recurrence is PI3Kα inhibition. PI3Kδ is an already approved therapeutic target in chronic lymphocytic leukemia, thus PI3Kδ may present a new therapeutically targetable opportunity for ER+BC recurrence. Elucidating the mechanism for acquired therapeutic resistance is an integral avenue for understanding how breast cancer progresses and improving the prognosis of ER+BC patients. Citation Format: Garhett Wyatt, Rachel Steinmetz, Traci Lyons, Weston Porter. LOSS OF SINGLEMINDED 2S RESULTS IN A PI3K SUBUNIT SWITCH WHICH DRIVES THERAPEUTIC RESISTANCE IN ESTROGEN RECEPTOR POSITIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-08.

Targeting the NTSR2/TrkB oncogenic pathway in chronic lymphocytic leukemia

Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies.

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eμ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eμ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eμ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

Evaluation of the novel therapeutic anti-CCR7 antibody CAP-100 as an add-on therapy in chronic lymphocytic leukemia patients receiving venetoclax.

The Bruton's tyrosine kinase inhibitor ibrutinib and the B-cell lymphoma 2 anti-apoptotic protein inhibitor venetoclax provide high response rates in chronic lymphocytic leukemia (CLL). However, there is a growing number of patients that relapse after treatment or show refractory disease, thus new targets and agents are still needed. We have previously reported the chemokine receptor CCR7 as a relevant deregulated target in CLL and have developed CAP-100, a novel therapeutic anti-CCR7 antibody that is under evaluation for relapse/refractory CLL (NCT04704323). While CCR7 expression has been shown to be down-modulated in CLL patients treated with ibrutinib, whether venetoclax acts in a similar manner remains unaddressed. Here, we aimed to document the impact of venetoclax on CCR7 expression in CLL cells, as well as on the pre-clinical activity of CAP-100. To this end, we addressed CCR7 expression by flow cytometry and the antibody efficacy by means of in vitro chemotactic and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Our data indicate that venetoclax treatment did not significantly modify CCR7 expression pattern nor CAP-100 mechanisms of action. Together, these findings support CAP-100 as an adjuvant therapy to venetoclax that may introduce additional modes of action in CLL therapy.

Significance of PPARA as a Treatment Target for Chronic Lymphocytic Leukemia.

Peroxisome proliferator-activated receptor alpha (PPARA) has been suggested as a therapeutic target for chronic lymphocytic leukemia (CLL). However, the underlying molecular mechanism remains largely unclear. In this study, we analyzed DNA next-generation sequencing (NGS) data and clinical information from 86 CLL patients to identify gene markers related to treatment-free survival (TFS) length. We then constructed a genetic network that includes CLL promoters, treatment targets, and TFS-related marker genes. To assess the significance of PPARA within the network, we utilized degree centrality (DC) and pathway enrichment score (EScore). Clinical and NGS data revealed 10 TFS length-related gene markers, including RPS15, FOXO1, FBXW7, KMT2A, NOTCH1, GNA12, EGR2, GNA13, KDM6A, and ATM. Through literature data mining, 83 genes were identified as CLL upstream promoters and treatment targets. Among them, PPARA exhibited a stronger connection to CLL and TFS-related gene markers, as evidenced by its ranking at No. 13 based on DC, compared to most of the other promoters (>84%). Additionally, PPARA co-functions with 70 out of 92 in-network genes in various functional pathways/gene groups related to CLL pathology, such as regulation of cell adhesion, inflammation, reactive oxygen species, and cell differentiation. Based on our findings, PPARA is considered one of the critical genes within a large genetic network that influences the prognosis and TFS of CLL through multiple pathogenic pathways.

Ezrin Is a Potential Druggable Target in Chronic Lymphocytic Leukemia

Despite development in therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or are refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors, but its role in hematological neoplasms is still poorly explored. There is evidence that EZR plays a key function in cell survival and activation of BCR-mediated signaling in B-cell lymphomas. Here, we uncover the expression of EZR in samples from CLL and healthy donors, and the cellular and molecular effects of the NSC305787, a pharmacological EZR inhibitor, in CLL cellular models. For gene expression studies, EZR mRNA expression data from healthy donors (normal lymphocytes, n = 11) and CLL patients (n = 103) samples were derived from the publicly accessible AmaZonia! database 2008 (discovery cohort). Purified CD19+ cells from peripheral blood samples were collected from 56 CLL patients and 10 age-matched healthy donors from the University Hospital of the Medical School of Ribeirão Preto (validation cohort). For functional and molecular assays, peripheral blood mononuclear cells of 24 CLL patients from the Instituto do Câncer do Estado de São Paulo were used. All samples were obtained after informed consent and protocol approved by the Ethical Committee of the Institution. For functional transcriptomics, RNA-seq data published by Landau et al. were obtained from cBioPortal, and all transcripts were pre-ranked according to their differential expression by comparing samples with high versus low EZR expression using limma-voom package in Galaxy and used for gene set enrichment analysis (GSEA). The MEC-1 cell line was cultured following the recommendations of DSMZ. PBMCs from CLL patients were cultured in RPMI Media 1640 and 10% autologous serum. NSC305787 was used as an EZR inhibitor. Cell viability was determined by MTT assay, apoptosis rates were determined by annexin V/PI labeling and flow cytometry, DNA content was performed by PI labeling and flow cytometry, protein expression was determined by Western Blot assay, and gene expression was evaluated by quantitative PCR. Statistical analyses were performed using GraphPad Prism 8; Mann-Whitney test, Student's t-test, or ANOVA and Bonferroni post-test were used for measurable factors, as appropriate. A value of p < 0.05 was considered statistically significant. EZR was highly expressed in lymphocytes derived from CLL patients (discovery cohort: p = 0.003; validation cohort: p = 0.005). GSEA indicated that high EZR expression was positively associated with relevant signaling pathways associated with CLL development and progression, including p53, PI3K/AKT/mTOR, NFkB, and MAPK (all p < 0.05 and FDR q < 0.05). Of note, pharmacological inhibition of EZR had remarkable antineoplastic effects in primary cells derived from CLL patients, corroborating the potential relevance of this target in disease maintenance. In a CLL cell line, EZR inhibition reduced viability, clonogenicity, cell cycle progression, and induced apoptosis (all p < 0.05). Pharmacological EZR inhibition also reduced activation of the ERK, S6RP, and NFkB, indicating that EZR not only associates, but participates in the activation of these signaling pathways in CLL. In conclusion, our results indicate that EZR is high expressed in CLL and associated with molecular signatures relevant to the development and maintenance of the disease. Pharmacological EZR inhibitions attenuate various cellular and molecular behaviors associated with high risk in CLL and may present a new class of drugs to expand the treatment repertoire of this disease. Supported by: FAPESP, CNPQ, and CAPES.

Tumor Heterogeneity-Based Sscr-Score Predicts Prognosis and Reveals Mechanism of SORL1 in Restraining CLL Progression

Introduction Complex heterogeneity is a hallmark of chronic lymphocytic leukemia (CLL) that challenges oncological research and optimal treatment regimens. Although genetic markers with prognostic values have been developed for risk assessment in CLL, the existing prognostic models are far from satisfaction. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to illustrate the genetic heterogeneity and cell differentiation status at the single-cell level. A prognostic model associated with CLL cell differentiation status was established to refine the risk assessment of CLL patients. Methods Primary CLL cells from 90 patients and CD19+ B cells from 20 healthy donors were collected with informed consents according to the Declaration of Helsinki. Expression levels of SORL1 mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Cell viability was measured by the CCK-8 assay. Cell cycle and apoptosis were analyzed by PI and Annexin V-PE/7AAD staining. Fatty acid oxidation rate was assessed with Fatty Acid Oxidation Complete Assay Kit. Red oil O staining was performed to evaluate the lipid deposition in CLL cells. Single cell sequencing data were analyzed by the Seurat package and Monocle package in R4.0.5. Results The scRNA-seq profile revealed remarkable intra- and inter-tumoral heterogeneity of CLL. According to the differentiation status, this study reconstructed the evolutionary history of CLL cells, which reflected the molecular classification and risk subgroups of CLL. At the key point of cell differentiation, CLL cells were divided into two subsets with progressive and indolent genetic signatures respectively. Genes differentially expressed between aggressive and indolent subsets were identified. Through univariate and multivariate analysis, four genes (SORL1, SGK1, CYBB, RPL22L1) with significant prognostic values were selected to construct a risk model named SSCR-Score. The risk score was calculated and validated in two independent validation cohorts (Fig 1A). SSCR-Score has revealed the positive prognostic value of SORL1 in CLL. Increased expression of SORL1 was associated with prolonged overall survival and improved time to first treatment in CLL. Among patients with ZAP70 (-) or IGHV mutated status, the expression levels of SORL1 were up-regulated. Although increased expression of SORL1 was associated with favorable prognosis, the biological mechanism of SORL1 still remained ill-defined. This study demonstrated that overexpression of SORL1 restrained cell proliferation, induced cell apoptosis and G2/M cell cycle arrest in CLL cells. In accordance with the RNA sequencing profile, overexpression of SORL1 suppressed abnormal lipid reposition and fatty acid oxidation in CLL cells. On a molecular level, SORL1 down-regulated the expression of lipid-metabolism related genes, including lipoprotein lipase (LPL), carnitine palmitoyltransferase 1A (CPT1A) and carnitine palmitoyltransferase 2 (CPT2). To illustrate the SORL1-mediated regulation of LPL, immunofluorescence staining was performed. Results showed that SORL1 accelerated the degradation of LPL. After the inhibition of lysosome function, accelerated degradation of LPL was not observed in CLL cells overexpressing SORL1. Rescue experiments substantiated that SORL1 impeded progression of CLL through downregulating the activity of LPL which promoted lipid metabolism. Immuno-coprecipitation experiment showed that anti-SORL1 antibody precipitated LPL protein. Additionally, 6-Shohaol induced overexpression of SORL1 in CLL cells. It exhibited potent anti-CLL efficacy by suppressing cell proliferation, promoting cell apoptosis and G2/M cell cycle arrest in CLL cells (Fig 1B). Conclusion CLL presented remarkable heterogeneity which reflected different biological subclasses and risk groups. Through identifying genes differentially expressed between aggressive and indolent cell subsets, a novel prognostic model named SSCR-Score was developed to optimize risk stratification of CLL patients. Moreover, SORL1 restrained progression of CLL by inhibiting lipid metabolism regulated by LPL in CLL cells. SORL1 may serve as a potential strategy for the development of novel therapeutic target in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ezrin is highly expressed and a druggable target in chronic lymphocytic leukemia

AimsDespite the development of therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors and plays a key role in cell survival and BCR-mediated signaling activation in B-cell lymphomas. However, its role in hematological neoplasms remains poorly explored. Main methodsThe present study assessed EZR expression in samples from CLL patients and healthy donors and evaluated the cellular and molecular effects of a pharmacological EZR inhibitor, NSC305787, in CLL cellular models. Key findingsEZR was highly expressed and positively associated with relevant signaling pathways related to CLL development and progression, including TP53, PI3K/AKT/mTOR, NF-κB, and MAPK. NSC305787 reduced viability, clonogenicity, and cell cycle progression and induced apoptosis in CLL cells. Pharmacological EZR inhibition also attenuated ERK, S6RP, and NF-κB activation, indicating that EZR not only associates with but also activates these signaling pathways in CLL. Ex vivo assays revealed that the EZR inhibition-induced cell viability reduction was independent of molecular risk and the Binet stage. SignificanceOur study provides insights into EZR as a pharmacological target in CLL, shedding light on a novel strategy for treating this disease.

CXCR4 and CD74 together enhance cell survival in response to macrophage migration-inhibitory factor in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lymphocytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single-pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co- and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n=10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in combination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL.

Integrin Signaling Shaping BTK-Inhibitor Resistance.

Integrins are adhesion molecules that function as anchors in retaining tumor cells in supportive tissues and facilitating metastasis. Beta1 integrins are known to contribute to cell adhesion-mediated drug resistance in cancer. Very late antigen-4 (VLA-4), a CD49d/CD29 heterodimer, is a beta1 integrin implicated in therapy resistance in both solid tumors and haematological malignancies such as chronic lymphocytic leukemia (CLL). A complex inside-out signaling mechanism activates VLA-4, which might include several therapeutic targets for CLL. Treatment regimens for this disease have recently shifted towards novel agents targeting BCR signaling. Bruton’s tyrosine kinase (BTK) is a component of B cell receptor signaling and BTK inhibitors such as ibrutinib are highly successful; however, their limitations include indefinite drug administration, the development of therapy resistance, and toxicities. VLA-4 might be activated independently of BTK, resulting in an ongoing interaction of CD49d-expressing leukemic cells with their surrounding tissue, which may reduce the success of therapy with BTK inhibitors and increases the need for alternative therapies. In this context, we discuss the inside-out signaling cascade culminating in VLA-4 activation, consider the advantages and disadvantages of BTK inhibitors in CLL and elucidate the mechanisms behind cell adhesion-mediated drug resistance.

P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES

Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity. Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem. Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE®) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, ‘CAM161519’, utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) ‘CAM16’, a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image). Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells. Image:Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products.

HIF-PH Encoded by EGLN1 Is a Potential Therapeutic Target for Chronic Lymphocytic Leukemia.

Owing to the recent emergence of drug resistance to Bruton’s tyrosine kinase inhibitors (BTK) in chronic lymphocytic leukemia (CLL) treatment, it is crucial to identify alternative therapeutic targets. Therefore, we aimed to identify therapeutic options for CLL besides BTK. We identified that HIF1A expression was higher in CLL patients than in controls, which may suggest good prognosis. We used a lentiviral knockdown of EGLN1 (encoding hypoxia-inducible factor prolyl hydroxylase [HIF-PH]) and found that the growth of MEC-1 cells slowed in the knockdown group. Treatment of CLL cell lines MEC-1 and HG3 with the HIF-PH inhibitor molidustat showed that molidustat could induce apoptosis in a concentration-dependent manner in CLL cells and had low cytotoxicity at this concentration. CXCR4, HIF1A, SLC2AI, and VEGF, the downstream molecules of the HIF pathway, were upregulated after molidustat treatment. Western blotting results indicated that molidustat increased HIF1A expression in CLL cell lines and cells from CLL patients, and sequencing/quantitative PCR analysis demonstrated that the ribosome biogenesis pathway was inhibited in MEC-1 cells after molidustat treatment. We further identified synergistic cytotoxicity of molidustat in combination with ibrutinib on the MEC-1 and HG3 cell lines at certain concentrations. Therefore, molidustat is a potential therapeutic option for CLL.

Preclinical activity of irreversible Menin inhibitor, BMF-219, in chronic lymphocytic leukemia.

7541 Background: Menin is a scaffold protein that drives oncogenic function through transcriptional modulation directed by its various cofactors. A previous report demonstrated that menin regulates a distinct set of gene targets independent of its function with the MLL proteins in hematopoiesis and is essential for B-cell maturation (Li et al. Blood.2013;122(12):2039-46.). Chronic Lymphocytic Leukemia (CLL) is a disease of malignant B lymphocytes, for which standard-of-care agents are generally well tolerated; however, CLL patients with certain genetic backgrounds demonstrate inferior outcomes to these regimens. A major driving feature of CLL is overexpression of the anti-apoptotic marker, BCL2. We previously reported the ability of BMF-219, a selective, irreversible menin inhibitor, to downregulate the expression of BCL2 in acute leukemia cells. Additionally, we have reported the synergy of BCL2-targeted agent, venetoclax, with BMF-219 in potent cell killing of diffuse large B-cell lymphoma (DLBCL) preclinical models, prompting our exploration of BMF-219 activity in CLL. Here, we provide the first preclinical evidence for menin as a therapeutic target in CLL, by demonstrating high potency of BMF-219 against a diverse collection of CLL patient specimens. Methods: A comprehensive panel of CLL samples isolated from patients with Rai Stage 1 to 3 disease, including relapsed or refractory disease, were cultured ex vivo in the presence of BMF-219 or clinical reversible menin inhibitors to assess the antileukemic activity of the compounds. Samples were analyzed for differential gene expression of select targets in response to treatment. Results: BMF-219 demonstrated high potency, achieving &gt; 98% cell lethality at 1 µM exposure in all patient samples tested, with IC50 values in the range of 0.1 to 0.38 µM. Specimens isolated from patients with clinical profiles of high-risk genetic backgrounds associated with inferior outcomes to standard therapy, such as mutations in TP53 and NOTCH1, and chromosomal aberrations such as del(13q), trisomy 12 and complex karyotype, exhibited high sensitivity to BMF-219 treatment. BMF-219 was also highly effective against patient samples with clinical profiles of resistance to bendamustine, ibrutinib and venetoclax therapy. In comparison, clinical reversible menin inhibitors demonstrated no significant activity across all patient samples tested, with incalculable IC50 values and &lt; 15% reduction in cell viability at 1 µM exposure. Expression of select target genes in treated CLL cell lines was explored and will be reported. Conclusions: Collectively, our data demonstrate the potent preclinical activity of BMF-219 against CLL patient specimens harboring various mutational and cytogenetic backgrounds, including categories of high-risk. These data highlight the unique potential of irreversible menin inhibition as a novel therapeutic option for patients with CLL.

Evaluation of allogeneic and autologous membrane-bound IL-21–expanded NK cells for chronic lymphocytic leukemia therapy

AbstractSuccesses with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21–expanded NK cells. Collectively, these data support development of mbIL-21–expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Elucidation of Focal Adhesion Kinase as a Modulator of Migration and Invasion and as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia.

Simple SummaryDespite the successful introduction of targeted therapies, Chronic Lymphocytic Leukemia (CLL) remains incurable. This is thought to be partially due to the pro-survival and anti-apoptotic signaling that CLL cells receive from the lymph node microenvironment. Therefore, inhibition of CLL migration into the lymph nodes is an attractive therapeutic option. Here, our aim was to gain a further understanding of what transcriptomic and miRNomic changes drive CLL migration and, from this, select promising therapeutic targets. We identified focal adhesion kinase (FAK) as one such potential target and demonstrated that inhibition of FAK in primary CLL samples effectively reduces both CXCL12 induced migration and invasion in vitro. Successful inhibition of CLL migration could increase the sensitivity of CLL cells to the currently used targeted therapeutics and therefore improve patient outcomes.The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation.

The indoleamine 2,3-dioxygenase 1 (IDO1) metabolic circuitry, comprising the first tryptophan (Trp) catabolite L-kynurenine (Kyn) and the aryl hydrocarbon receptor (AHR), has emerged as a mechanism of cancer immune evasion. Here, we investigated the functional role of the IDO1/Kyn/AHR axis in chronic lymphocytic leukemia (CLL). Our data show that CLL cells expressed an active form of the IDO1 enzyme and microenvironmental stimuli can positively modulate its expression. Interferon (IFN)-γ induces IDO1 expression through the Jak/STAT1 pathway and mediates Kyn production concomitantly with Trp consumption in CLL-conditioned media, while INCB018424 (ruxolitinib), a JAK1/2 inhibitor, impaired both effects. To characterize the involvement of IDO1 in leukemic cell maintenance, we overexpressed IDO1 by vector transfection measuring enhanced resistance to spontaneous apoptosis. IDO1 pro-survival influence was confirmed by treating CLL cells with Kyn, which mediated the increase of induced myeloid leukemia cell differentiation protein (MCL1). Conversely, AHR silencing or its blockade via CH-223191 improved the apoptosis of leukemic clones and mitigated MCL1 expression. Moreover, Kyn-treated CLL cells are less affected by the pro-apoptotic effect of ABT-199 (venetoclax), while CH-223191 showed synergistic/additive cytotoxicity with this drug. Lastly, targeting directly MCL1 in CLL cells with AMG-176, we abrogate the pro-survival effect of Kyn. In conclusion, our data identify IDO1/Kyn/AHR signaling as a new therapeutic target for CLL, describing for the first time its role in CLL pathobiology.

Identification of CD105 (endoglin) as novel risk marker in CLL

Several genetic and clinical markers are established as prognostic factors in chronic lymphocytic leukemia (CLL). However, additional markers are needed for risk stratification. Flow cytometric analysis is a mainstay of CLL diagnostics, thus identification of novel prognostic surface markers can improve risk assessment without increasing burden for patients and physicians. Furthermore, surface molecules preferentially expressed in high-risk cases could serve as therapeutic targets for immunotherapy. CD105 (endoglin) is a TGF-beta coreceptor and activates endothelial cells in healthy tissues and cancer. In addition, it is expressed on healthy hematopoietic precursors as well as lymphoid and myeloid leukemias. In acute myeloid leukemia (AML), a CD105 antibody is successfully applied in clinical studies. In CLL, mRNA expression of the CD105 gene ENG reportedly correlates with other risk factors but failed to show significant correlation with overall survival. However, CD105 protein expression in CLL has never been studied. We here analyzed CD105 surface expression on CLL cells from 71 patients by flow cytometry and report for the first time that substantial levels of CD105 are detectable on CLL cells in 70.4% of patients. Using receiver operating characteristics, we established a cutoff of 5.99% positive cells to distinguish between low and high CD105 levels, the latter correlating with decreased time to first treatment and overall survival. High CD105 expression further correlates with CD38 expression. Our study identified membrane expression of CD105 as a potential risk marker and therapeutic target in high-risk CLL. However, multivariant analyses of large cohorts should be performed in confirmatory studies.

Virtual Screening of Adenylate Kinase 3 Inhibitors Employing Pharmacophoric Model, Molecular Docking, and Molecular Dynamics Simulations as Potential Therapeutic Target in Chronic Lymphocytic Leukemia

Adenylate kinase 3 (AK3) is an enzyme located in the mitochondrial matrix involved in purine homeostasis. This protein has been considered a potential therapeutic target in chronic lymphocytic leukemia (CLL), because the silencing of the AK3 gene has inhibited cell growth in CLL in vitro models. This study aimed to design potential AK3 inhibitors by applying molecular modeling techniques. Through the mapping of AK3 binding sites, essential interaction fields for pharmacophore design were identified. Online libraries were virtually screened by using a pharmacophore model, and 6891 compounds exhibited the functional groups for interaction with the target. These compounds underwent molecular docking simulations through Surflex and GOLD programs. After visual inspection, we selected 13 compounds for pharmacokinetic properties toxicology prediction via admetSAR and Protox web servers. Finally, six compounds were chosen for further analysis.

Integrated Analyses of Competing Endogenous RNA Network Reveal Potential Therapeutic Targets in Chronic Lymphocytic Leukemia

IntroductionChronic lymphocytic leukemia (CLL) is a highly heterogeneous disease characterized by malignant clonal expansion of mature B lymphocytes. Competitive endogenous RNAs(ceRNAs) such as long noncoding RNAs (lncRNAs) and circular RNAs (circ RNAs) have miRNA response elements (MREs) and can bind to miRNAs to influence mRNA expression. An increasing number of studies have shown that the ceRNA network played an important role in the initiation and progression of tumors. However, the roles and functions of the ceRNA network in chronic lymphocytic leukemia (CLL) are still unclear. This study aims to explore the molecular mechanism of CLL and provide potential prognostic markers and therapeutic targets through the integrated analysis of the ceRNA network in CLL.MethodsThe expression profile of RNAs of CLL patients, CLL cell lines (MEC1 and EHEB) and healthy group were obtained by the illumina sequencing. R software was used for functional enrichment analysis. The data in the genome microarray map GSE22762 was used for survival analysis. The circRNA-miRNA-mRNA ceRNA networks were visualized by Cytoscape 3.7.2. The expression of the circRNA hsa_circ_0007675/hsa-miR-185-3p/TCF7L1 axis were verified by Quantitative real-time PCR and the correlation between hsa_circ_0007675 and TCF7L1 was analyzed.ResultsIn total, we identified 57 differentially expressed mRNAs (DEmRNAs), 1391 DElncRNAs, 335 DEmiRNAs and 2413 DEcircRNAs by comparing CLL patients with healthy donors. Meanwhile, 482 mRNAs, 6085 lncRNAs, 302 miRNAs and 1847 circRNAs were explored differently expressed between CLL cell lines and healthy donors.GO analysis results showed that the functions of differentially expressed genes (DEGs) between CLL patients and control are mainly enriched in sequence−specific DNA binding, chromatin and gene expression (Figure 1A) while between CLL cell lines and control they were mainly enriched in oxidoreductase activity, ribosomal subunit and lipid metabolism (Figure 1C). KEGG pathway analysis revealed that the DEGs between CLL patients and control were mainly enriched in Notch signaling pathway, JAK-STAT signaling pathway and cGMP-PKG signaling pathway (Figure 1B). Meanwhile between CLL cell lines and control, DEGs were mainly enriched in mTOR signaling pathway, cell cycle and p53 signaling pathway (Figure 1D).The survival analyses showed that 15 DEGs (INIP, IL3RA, CHD1, NLRP12, IL20RB, HNRNPC, B3GALT4, SIT1, ACOT8, PCLAF, C19orf18, SELENOS, OR7A17, PCDH7, PHGDH) were significantly differentially expressed in the survival analyses. The overall survival of the high expression group of INIP, IL3RA, CHD1, NLRP12, IL20RB and HNRNPC were higher than that of the low expression group (Figure 2A-F) while the overall survival of the low expression group of B3GALT4, SIT1, ACOT8, PCLAF, C19orf18, SELENOS, OR7A17, PCDH7 and PHGDH were higher than that of the high expression group (Figure 2G-O).The ceRNA network were built by Cytoscape3.7.2. In total, 11 mRNA nodes, 19 miRNA nodes, 251 circRNA nodes were identified as differentially expressed profiles between CLL patients and control (Figure 3A). We verified the circRNA hsa_circ_0007675/hsa-miR-185-3p/TCF7L1 axis. Compared with normal people, the expression of TCF7L1 and hsa_circ_0007675 in patient specimens were significantly increased (p<0.05; Figure 3B, D) whereas the expression of hsa-miR-185-3p was downregulated (p<0.05; Figure 3C). TCF7L1 and hsa_circ_0007675 were positively correlated (p＜0.001, R=0.7834; Figure 3E). The correlation analysis of TCF7L1 and other genes were shown in Figure 3F. The interaction mechanism between them is that hsa_circ_0007675 can sponge hsa-miR-185-3p, thereby inhibiting the inhibitory effect of hsa-miR-185-3p on TCF7L1 and finally upregulate the expression of TCF7L1(Figure 3G).ConclusionsIn this study, we identified the expression profile of RNAs in CLL patients and CLL cell lines. Functional enrichment analysis and survival analysis revealed the potential functions of DEGs. The ceRNA network we established can help to further understand the pathogenesis of CLL and provide potential prognostic biomarkers and novel therapeutic targets.Keywords: Chronic lymphocytic leukemia; Competing endogenous RNA; Non-coding RNAs; Prognostic biomarkers; Therapeutic targets [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Venetoclax-Resistant CLL By Bcl-XL Degradation

Targeting Venetoclax-Resistant CLL By Bcl-XL Degradation