Proliferation Of Chronic Lymphocytic Leukemia Cells Research Articles

Apoliprotein E-mediated ferroptosis controls cellular proliferation in chronic lymphocytic leukemia.

Unraveling vulnerabilities in chronic lymphocytic leukemia (CLL) represents a key approach to understand molecular basis for its indolence and a path toward developing tailored therapeutic approaches. In this study, we found that CLL cells are particularly sensitive to the inhibitory action of abundant serum protein, apolipoprotein E (ApoE). Physiological concentrations of ApoE affect CLL cell viability and inhibit CD40-driven proliferation. Transcriptomics of ApoE-treated CLL cells revealed a signature of redox and metal disbalance which prompted us to explore the underlying mechanism of cell death. We discover, on one hand, that ApoE treatment of CLL cells induces lipid peroxidation and ferroptosis. On the other hand, we find that ApoE is a copper-binding protein and that intracellular copper regulates ApoE toxicity. ApoE regulation tends to be lost in aggressive CLL. CLL cells from patients with high leukocyte counts are less sensitive to ApoE inhibition, while resistance to ApoE is possible in transformed CLL cells from patients with Richter syndrome (RS). Nevertheless, both aggressive CLL and RS cells maintain sensitivity to drug-induced ferroptosis. Our findings suggest a natural suppression axis that mediates ferroptotic disruption of CLL cell proliferation, building up the rationale for choosing ferroptosis as a therapeutic target in CLL and RS.

The Novel Anti-Cancer Agent, SpiD3, Is Cytotoxic in CLL Cells Resistant to Ibrutinib or Venetoclax.

B-cell receptor (BCR) signaling is a central driver in chronic lymphocytic leukemia (CLL), along with the activation of pro-survival pathways (e.g., NF-κB) and aberrant anti-apoptotic mechanisms (e.g., BCL2) culminating to CLL cell survival and drug resistance. Front-line targeted therapies such as ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor) have radically improved CLL management. Yet, persisting CLL cells lead to relapse in ~20% of patients, signifying the unmet need of inhibitor-resistant refractory CLL. SpiD3 is a novel spirocyclic dimer of analog 19 that displays NF-κB inhibitory activity and preclinical anti-cancer properties. Recently, we have shown that SpiD3 inhibits CLL cell proliferation and induces cytotoxicity by promoting futile activation of the unfolded protein response (UPR) pathway and generation of reactive oxygen species (ROS), resulting in the inhibition of protein synthesis in CLL cells. We performed RNA-sequencing using CLL cells rendered resistant to ibrutinib and venetoclax to explore potential vulnerabilities in inhibitor-resistant and SpiD3-treated CLL cells. The transcriptomic analysis of ibrutinib- or venetoclax-resistant CLL cell lines revealed ferroptosis, UPR signaling, and oxidative stress to be among the top pathways modulated by SpiD3 treatment. By examining SpiD3-induced protein aggregation, ROS production, and ferroptosis in inhibitor-resistant CLL cells, our findings demonstrate cytotoxicity following SpiD3 treatment in cell lines resistant to current front-line CLL therapeutics. Our results substantiate the development of SpiD3 as a novel therapeutic agent for relapsed/refractory CLL disease.

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

A Review of Resistance Mechanisms to Bruton's Kinase Inhibitors in Chronic Lymphocytic Leukemia.

Bruton's Tyrosine Kinase (BTK) inhibitors have become one of the most vital drugs in the therapy of chronic lymphocytic leukemia (CLL). Inactivation of BTK disrupts the B-cell antigen receptor (BCR) signaling pathway, which leads to the inhibition of the proliferation and survival of CLL cells. BTK inhibitors (BTKi) are established as leading drugs in the treatment of both treatment-naïve (TN) and relapsed or refractory (R/R) CLL. Furthermore, BTKi demonstrate outstanding efficacy in high-risk CLL, including patients with chromosome 17p deletion, TP53 mutations, and unmutated status of the immunoglobulin heavy-chain variable region (IGHV) gene. Ibrutinib is the first-in-class BTKi which has changed the treatment landscape of CLL. Over the last few years, novel, covalent (acalabrutinib, zanubrutinib), and non-covalent (pirtobrutinib) BTKi have been approved for the treatment of CLL. Unfortunately, continuous therapy with BTKi contributes to the acquisition of secondary resistance leading to clinical relapse. In recent years, it has been demonstrated that the predominant mechanisms of resistance to BTKi are mutations in BTK or phospholipase Cγ2 (PLCG2). Some differences in the mechanisms of resistance to covalent BTKi have been identified despite their similar mechanism of action. Moreover, novel mutations resulting in resistance to non-covalent BTKi have been recently suggested. This article summarizes the clinical efficacy and the latest data regarding resistance to all of the registered BTKi.

IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

Abstract P01: Impact of Germline and Somatic ATM Variants in Chronic Lymphocytic Leukemia (CLL): Clinical Implications and Response to PARP Inhibition

Abstract Background: Next-generation sequencing has revealed numerous Variants of Unknown Significance (VUS) in the ATM gene, important in DNA repair and chronic lymphocytic leukemia (CLL) pathogenesis. This study evaluates the effect of rare ATM germline variants, including VUS, on features of CLL, ATM kinase activity, and response to PARP inhibition. Methods:ATM variants were detected in 885 CLL patients (327 DFCI; 276 German CLL Study Group; 282 ICGC) using DeepVariant, and compared to 5310 ancestry-matched controls. Concurrently, 278 CLL patients with ATM aberrations from clinical sequencing were retrospectively analyzed, annotated by population allele frequency (JCO 2023, Lampson et al.). ATM kinase activity was measured by ATM and KAP1 phosphorylation following gamma-irradiation in primary CLL samples. The impact of talazoparib, a PARP inhibitor, on ATM aberrant and wild-type CLL cell proliferation was also assessed. Results: Among the 885 CLL cases, 130 ATM germline variants were identified by DeepVariant, including 87 missense variants. The top 5 most frequent rare germline missense variants with high odds ratio for association with CLL were ATM p.L2332P (AF cases vs controls, 0.328% vs 0.0188%, OR 17.46), p.L2307F (OR 16.88), p.F763L (OR 18.89), p.Y1442H (OR 35.39) and p.S99G (OR 16.28). In our retrospective clinical cohort, we identified 294 ATM variants with 146 unique variants. Our previously proposed algorithm for classifying germline vs somatic identified 74 germline, with 70 missense, and 72 somatic. Phosphorylation of ATM and KAP1 (pATM, pKAP1) in response to IR was reduced in 13 patients assessed with germline ATM variants (9 rare and 4 non-rare; ATM variant alone group) compared to those without any somatic or germline ATM aberrancy (WT group) (P<0.01). Phosphorylation levels were also significantly lower in 17 patients with rare germline ATM variants with concurrent del(11q) (del11q + ATM germline variant group) compared to 15 with del(11q) alone (del 11q group) (P<0.01). We obtained similar results from Western blot analysis. To assess the sensitivity of ATM deficient cells to talazoparib, we compared proliferation inhibition across groups cocultured with talazoparib for 14 days. The del11q with ATM-somatic group exhibited significantly greater sensitivity to talazoparib at lower dosages compared to ATM WT (talazoparib 0.1 μM, P<0.05; 0.5μM, P=0.098). The monoallelic ATM deficient groups (del11q alone or ATM-germline alone) tended to have lower proliferation, but not significantly. Higher concentrations of talazoparib (2.5μM) strongly inhibited proliferation of all CLLs including WT. Conclusion: This study highlights the prevalence of rare ATM germline variants in CLL and their impact on ATM function in CLL. Our findings also suggest that these variants affect the DNA damage response in primary CLL cells and that ATM biallelic deficiency influences sensitivity to PARP inhibitors. Our evidence could lead to more personalized therapeutic strategies for CLL patients with distinct ATM aberrancies. Citation Format: Kiyomi Mashima, Nicholas Moore, Mariia Mikhaleva, Anna Petráčková, Samantha Shupe, Stacey M Fernandes, Amaro Taylor-Weiner, Riaz Gillani, Hoyin Chu, Seunghun Han, Sabrina Camp, Eric Kofman, Gad Getz, Catherine J. Wu, Svitlana Tyekucheva, Matthew S Davids, Eliezer Mendel Van Allen, Saud AlDubayan, Jennifer R Brown. Impact of Germline and Somatic ATM Variants in Chronic Lymphocytic Leukemia (CLL): Clinical Implications and Response to PARP Inhibition [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P01.

MS-553 Therapy Inhibits BCR Signaling and Increases Survival Dependence on BCL-2 in BTK-Inhibitor Resistant CLL

B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL. We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

Transcriptomic Analyses Indicate That Pre-Leukemic B Cells from People with Monoclonal B Lymphocytosis Are Surprisingly Much Less Active Than Normal B Cells from People with Monoclonal B Lymphocytosis and from Healthy Donors

Introduction. Chronic lymphocytic leukemia (CLL) follows a stepwise developmental process, beginning in a specific group of normal B lymphocytes that undergo clonal expansion. These clonal expansions, known as monoclonal B lymphocytosis (MBL), are considered to be essential precursors of CLL based on immunologic, genetic, and epidemiologic studies. Since MBL represents the overabundance of a single CD5 + B cell, we tested how different the transcriptomes of MBL cells were from normal B cells (NBC) in people with MBL (NBC-MBL) and NBC in healthy donors (NBC-HD), expecting to identify activations pathways in the MBL cells that would explain clonal expansion. Methods. We collected and sorted specific cell populations from 13 patients with monoclonal B lymphocytosis (MBL): CD5 +CD19 +CD20 dimIgL-restricted MBL cells and CD5 -CD19 +CD20 + NBC. Additionally, we obtained CD5 -CD19 +CD20 + NBC from 13 healthy donors (NBC-HD). RNA was extracted and sequenced using SMART-Seq v4 Ultra Low Input and HiSeq platform. DESeq2 was used to analyze RNA-seq data. To identify genes with differential expression between these populations, the transcriptomes of MBL cells were compared to NBC from HD, employing a cutoff of P<0.05 and Log2fc ratio >± 1.5. We also used paired sample t-tests to compare 13 MBL cells to NBC from the same MBL patients. Significantly differentially expressed genes (DEG) resulting from these analyses were then subjected to Ingenuity Pathway Analysis (IPA). Results. When comparing the gene profiles of 13 MBL B cells to 13 NBC-HD cells, we identified 2695 DEG. Next, we compared gene profiles between MBL B cells and NBC-MBL cells obtained from 13 MBL patients, identifying 4821 genes. Using IPA, down-regulated B Cell Receptor Signaling, Cytokine Signaling, and Tumor Microenvironment Pathway were identified in both analyses. Upon overlapping the genes from these two analyses, we found that 803 genes passed the cutoff in both analyses. IPA analysis showed that 14 signaling pathways, including IL-33, IL-17, and HIF1a, were significantly downregulated in MBL cells. Moreover, the negative signaling pathway (Role of p14/p19ARF in Tumor Suppression) was upregulated in MBL cells. PDCD1 expression was significant higher in MBL (Log2Fc 6.8, P<0.0001) and 11 PDCD1 target genes were significantly regulated in MBL cells. IL-6 (Log2Fc -5.4, P<0.0001) expression was significantly lower in MBL and 47 IL-6 target genes were significantly regulated in MBL cells. Surprisingly, genes related to cell activation, proliferation and movement were largely downregulated in MBL cells. There were 2052 genes unique to the analysis MBL vs NBC-HD, IPA analysis showed several T-cell related signaling pathways e.g., T cell Exhaustion Signaling Pathway and T Lymphocyte Apoptosis were upregulated. In the 4018 genes unique in the analysis MBL vs NB-MBL. IPA results showed all the signaling pathways were downregulated in MBL. Discussion. Surprisingly, our transcriptomic analyses indicate that MBL cells are much less active than NBC from people with MBL and from HD. Specifically, we found that many cytokine signaling pathways and genes related with cell activation, proliferation and movement are downregulated in MBL cells. Also, PDCD1 is highly expressed in MBL cells, and PDCD1 target genes are also significantly regulated in MBL cells. This is consistent with the immunosuppressed features of MBL cells since interaction between PD-1 on B cells and its ligands in the microenvironment negatively influence the survival, proliferation, and immune evasion of CLL and MBL cells. Collectively, although functional studies are needed, these transcriptomic data strongly suggest that MBL cells are not in an activated state and are less active than even the NBC from the same person with MBL.

The Unique BCR Inhibiting Properties of BMS-986205 in Chronic Lymphocytic Cells

B cell receptor (BCR) signaling is recognised as a central pathway in the pathogenesis of chronic lymphocytic leukaemia (CLL), the most common leukemia in the western world. In CLL, the BCR pathway is activated by antigens in the tissue microenvironment to promote the maintenance and expansion of leukemic cells. The impressive inhibition of clonal expansion of CLL cells and the disease control achieved with tyrosine kinase inhibitors that block BCR signaling provided further evidence of the role of BCR in CLL. Indoleamine 2,3-dioxygenase 1 (IDO1) is the inducible and rate-limiting enzyme involved in the catabolism of tryptophan to kynurenine. It was discovered as a modulator of the innate immune response during infection. Subsequent discoveries have implicated IDO1 as a player in acquired immune tolerance. IDO1 activity has been identified in several types of cancer cells as well as in their tumor-surrounding microenvironment. We have previously investigated and characterized the impact of IDO1 expression in CLL. Our results show that CLL microenvironmental stimuli positively modulate IDO1 expression in leukemic cells and that increased IDO1 activity leads to sustained survival of malignant lymphocytes and impaired drug sensitivity through the autocrine/paracrine activation of the aryl hydrocarbon receptor by kynurenine (Atene CG et al. 2022). To assess the functional and clinical significance of IDO1 enzymatic activity in CLL, we used several inhibitors that are currently in late-stage clinical trials. Using BMS-986205 (BMS), an irreversible inhibitor of IDO1 with the best cell-based potency, in primary patient cells, we observed not only a reduction in kynurenine production due to enzymatic inhibition of IDO1, but also lower levels of IDO1 RNA and protein following BCR stimulation with α-IgM. As we have previously shown that BCR activation can induce IDO1 expression, we treated CLL cells with increasing doses of BMS for 3 hours and then added α-IgM to the culture. BMS is able to counteract the pro-survival effect of BCR triggering, leading to significant apoptosis of CLL cells, while the viability of CLL cells in the absence of external stimuli was not significantly modulated. Focusing on the complex signaling cascade activated by BCR engagement, we observed that BMS impaired the phosphorylation of upstream kinases such as SYK and BTK, but also CD79a, CD19 and BLNK, in addition to downstream effectors such as AKT. We therefore investigated which factors that negatively regulate the BCR pathway are modulated by BMS treatment. By analyzing both mRNA and protein levels, we measured a significant induction of the phosphatases PTPN6, PTPN22 and SHIP1. Since BMS also showed a downregulation of total SYK expression, we are analyzing the expression of the proto-oncogene CBL, an E3 ubiquitin ligase known to be involved in the negative regulation of SYK. Further studies should be undertaken to determine whether, in addition to its role as an IDO1 inhibitor, BMS may also have the ability to indirectly modulate and disrupt BCR signaling. Efforts are underway to identify the proteome-wide direct target of BMS in CLL cells. In conclusion, our limited and preliminary data suggest that BMS could be considered as a potential therapeutic strategy to target CLL cells with a double hit: by blocking the pro-survival effect of BCR signaling, one of the driving forces for CLL cell survival and proliferation, and by inhibiting IDO1/kynurenine action to restore sensitivity to key leukemia treatments.

CLL Cells within the Proliferation Centers of the Patient Lymph Nodes Are Enriched for Notch Signaling, Thus NOTCH a Viable Target for CLL Therapy

CLL cells within the Proliferation Centers of the Patient Lymph Nodes are enriched for Notch signaling, thus a Target for CLL Therapy. Shantaram S. Joshi, Michael Pitner, Matt Kling, Furqan Mehdi, Mamta Shukla, Ashima Shukla, Vipul Shukla, Nagendra Chaturvedi, Hamid Band , Javeed Iqbal The tissue microenvironment, particularly the lymph node (LN), plays a major role in regulating the leukemic progression and clinical heterogeneity of chronic lymphocytic leukemia (CLL). CLL cells proliferate in the patient lymph nodes, resulting in the formation of proliferation centers (PCs). The molecular basis of the CLL cell proliferation within PCs is not well known. Several reports have implicated the activated Notch signaling pathway in the pathogenesis of CLL. Using the IRF4 -/-Vh11 mouse model of CLL, we have previously shown that Notch signaling is indispensable for CLL development (Vipul Shukla et al, Oncotarget, 2016). Based on these studies, we hypothesized that CLL cell proliferation in the lymph node PCs involves activated NOTCH signaling. To this end, we have examined the expression levels of Notch signaling associated molecules namely IRF4, Notch 1, Notch 2, Nedd4, Fbxw7 and Itch1 on CLL cells within the patient lymph node proliferation centers (n=12) using immunohistochemistry and western blotting. Expression levels were scored based on the percentage of cells with positive staining: 5+ (>80%), 4+ (60-80%), 3+ (40-60%), 2+ (20-40%), 1+ (<20%) and 0 (No staining). IHC analyses showed that the expression levels of IRF4 to be 2+, Notch 1 was 4+, Notch 2 was -3 to 4+, Nedd4 was 1+, Fbxw7 was 3+ and ITCH1 was 1+. Western blotting of CLL cells from the lymph node proliferation centers (n=6) confirmed a similar pattern of expression of the Notch-associated genes. Together, these results validate the association of Notch signaling in CLL cells of the patient lymph node PCs similar to CLL cells from the IRF4 -/-Vh11 mouse model. Therefore, as a next logical step we treated the MEC-1 and OSU-CLL cells in culture and primary CLL cell from patients with NOTCH inhibitor RO4929097 (RO), a γ-secretase inhibitor for 24, 48 and 72 hours in vitro. There was a significant decrease in the expression NOTCH 1, NOTCH 2 in MEC-1, OSU-CLL and primary CLL cells determined using western blot technique. Also, we observed a significant decrease in proliferation of CLL cells from MEC-1 and OSU-CLL cell lines, following treatment with NOTCH inhibitor RO. In addition, NOTCH inhibitor combined with Bruton's Tyrosine Kinase (BTK) a member of the B-Cell Receptor signaling, showed a further significant decrease in the proliferation of CLL cells in culture. Together these experiments demonstrated that NOTCH targeting strategy is a viable therapy for aggressive CLL warranting further preclinical and clinical studies. This work was partially supported by bridge funding from the American Association of Hematology awarded to Dr. Runqing Lu, who passed away during the pursuit of this study.

M6A-Modified circTET2 Interacting with HNRNPC Regulates Fatty Acid Oxidation to Promote the Proliferation of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a hematological malignancy with high metabolic heterogeneity. N6-methyladenosine (m6A) modification plays an important role in metabolism through regulating circular RNAs (circRNAs). However, the underlying mechanism is not yet fully understood in CLL. Herein, an m6A scoring system and an m6A-related circRNA prognostic signature are established, and circTET2 as a potential prognostic biomarker for CLL is identified. The level of m6A modification is found to affect the transport of circTET2 out of the nucleus. By interacting with the RNA-binding protein (RBP) heterogeneous nuclear ribonucleoprotein C (HNRNPC), circTET2 regulates the stability of CPT1A and participates in the lipid metabolism and proliferation of CLL cells through mTORC1 signaling pathway. The mTOR inhibitor dactolisib and FAO inhibitor perhexiline exert a synergistic effect on CLL cells. In addition, the biogenesis of circTET2 can be affected by the splicing process and the RBPs RBMX and YTHDC1. CP028, a splicing inhibitor, modulates the expression of circTET2 and shows pronounced inhibitory effects. In summary, circTET2 plays an important role in the modulation of lipid metabolism and cell proliferation in CLL. This study demonstrates the clinical value of circTET2 as a prognostic indicator as well as provides novel insights in targeting treatment for CLL.

Expression and Activity of the NF-κB Subunits in Chronic Lymphocytic Leukaemia: A Role for RelB and Non-Canonical Signalling.

Canonical NF-κB signalling by p65 (RelA) confers chemo-resistance and poor survival in chronic lymphocytic leukaemia (CLL). The role of non-canonical NF-κB signalling (leading to RelB and p52 subunit activation) in CLL is less understood, but given its importance in other B-cell tumour types, we theorised that RelB and p52 may also contribute to the pathology of CLL. DNA binding activity of all five NF-kB subunits, p65, p50, RelB, p52, and c-Rel, was quantified using ELISA and correlated to ex vivo chemoresistance, CD40L-stimulated signalling (to mimic the lymph node microenvironment), and clinical data. Importantly, we show for the first time that high basal levels of RelB DNA binding correlate with nuclear RelB protein expression and are associated with del(11q), ATM dysfunction, unmutated IGHV genes, and shorter survival. High levels of nuclear p65 are prevalent in del(17p) cases (including treatment-naïve patients) and also correlate with the outcome. CD40L-stimulation resulted in rapid RelB activation, phosphorylation and processing of p100, and subsequent CLL cell proliferation. These data highlight a role for RelB in driving CLL cell tumour growth in a subset of patients and therefore strategies designed to inhibit non-canonical NF-κB signalling represent a novel approach that will have therapeutic benefit in CLL.

The Evolution of Therapies Targeting Bruton Tyrosine Kinase for the Treatment of Chronic Lymphocytic Leukaemia: Future Perspectives.

The development of inhibitors of Bruton tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL2) has resulted in a paradigm shift in the treatment of chronic lymphocytic leukaemia (CLL) over the last decade. Observations regarding the importance of B-cell receptor signalling for the survival and proliferation of CLL cells led to the development of the first-in-class BTK inhibitor (BTKi), ibrutinib, for the treatment of CLL. Despite being better tolerated than chemoimmunotherapy, ibrutinib does have side effects, some of which are due to the off-target inhibition of kinases other than BTK. As a result, more specific inhibitors of BTK were developed, such as acalabrutinib and zanubrutinib, which have demonstrated equivalent/enhanced efficacy and improved tolerability in large randomized clinical trials. Despite the increased specificity for BTK, side effects and treatment resistance remain therapeutic challenges. As these drugs all bind covalently to BTK, an alternative approach was to develop noncovalent inhibitors of BTK, including pirtobrutinib and nemtabrutinib. The alternative mechanisms of BTK-binding of these agents has the potential to overcome resistance mutations, something that has been borne out in early clinical trial data. A further step in the clinical development of BTK inhibition has been the introduction of BTK degraders, which remove BTK by ubiquitination and proteasomal degradation, in marked contrast to BTK inhibition. This article will review the evolution of BTK inhibition for CLL and offer future perspectives on the sequencing of an increasing number of different agents, and how this may be impacted on by mutations in BTK itself and other kinases.

Abstract 4933: Duvelisib eliminates CLL B Cells, impairs CLL-supporting cells, and overcomes ibrutinib resistance in a patient-derived xenograft model

Abstract Duvelisib (IPI-145), a first-in-class, oral, dual PI3K-δ,γ inhibitor, has been approved by the FDA for the treatment of patients with chronic lymphocytic leukemia (CLL), small lymphocytic and follicular lymphoma. Duvelisib potently inhibits PI3K-δ and PI3K-γ, thereby offering a novel therapeutic approach. Here we aimed to understand how duvelisib targets CLL cells and the tumor microenvironment (TME) and hence how it affects CLL, even with BTK-resistant disease. We first assessed the distinct contributions of PI3K-δ and PI3K-γ on CLL cell survival, proliferation, and migration in vitro by using PI3K-δ (PI3K-δi) or PI3K-γ (PI3K-γi) specific inhibitors in addition to duvelisib. We found a significant reduction in viable CLL cells after exposure to PI3K-δi and duvelisib. By measuring Ki67 and p-AKT levels, we found duvelisib and PI3K-δi more potently block CLL cell proliferation than PI3K-γi. PI3K-δi and duvelisib also significantly reduced the migration of CLL B cells into the spleen relative to control. In contrast, PI3K-γi inhibited T cell not B cell homing in vitro and in vivo. We then investigated the impact of single versus dual inhibitory agents on myeloid cells. Tumor-associated macrophages (TAMs) of the “M2 phenotype” contribute to a pro-tumor TME by preventing the induction of T cell mediated anti-tumor immunity. We expanded murine bone marrow derived monocytes and then polarized them to M2 macrophages, in the absence or presence of PI3Ki. At 100nM, PI3K-γi, but not PI3K-δi, significantly inhibited M2 polarization. Duvelisib significantly reduced Arg1 expression at doses equal to or above 10nM. While co-culturing leukemic B cells with M2-polarized murine macrophages increased CLL-cell survival, addition of duvelisib to such co-cultures significantly reduced CLL cell survival. Altogether, duvelisib sensitizes primary CLL cells to apoptosis and abrogates M2 cell-mediated CLL-cell survival. Finally, in a patient-derived xenograft mouse model (PDX), we demonstrated the essential roles of PI3K-δ and PI3K-γ in the CLL TME. Specifically, we found a more efficacious inhibition in CLL cell burden by dual inhibition of PI3K-δ,γ. Also, samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in PDX, irrespective of BTK mutations. In support of this, we identified an ibrutinib resistant CLL patient, with a clone exhibiting BTK and PLCγ2 mutations who responded immediately to single agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with subsequent modulation of T and myeloid cells. In conclusion, our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B cell numbers and T and myeloid cell pro-leukemia functions, supporting the use of duvelisib as a potentially valuable therapeutic intervention, including for patients refractory to BTKi. Citation Format: Shih-Shih Chen, Jacqueline C. Barrientos, Gerardo Ferrer, Priyadarshini Ravichandran, Michael Ibrahim, Yasmine Kieso, Jeffery L. Kutok, Marisa Peluso, Sujata Sharma, David T. Weaver, Jonathan A. Pachter, Kanti R. Rai, Nicholas Chiorazzi. Duvelisib eliminates CLL B Cells, impairs CLL-supporting cells, and overcomes ibrutinib resistance in a patient-derived xenograft model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4933.

Novel therapies and combinations in CLL refractory to BTK inhibitors and venetoclax.

Patients with chronic lymphocytic leukemia (CLL) refractory to covalent BTK and BCL2 inhibitors have a new unmet clinical need. Standard treatment options are able to obtain only limited and short-lasting disease control associated with reduced overall survival, and thus these patients have become ideal candidates for enrollment in clinical trials. Favorable results have been obtained with the use of noncovalent BTK inhibitors (roughly 70% overall response rate regardless of the actual resistance or intolerance to previous covalent BTK inhibitors) and anti-CD19 chimeric antigen receptor (CAR) T-cell therapy (with complete responses in up to 45% of cases and an undetectable measurable residual disease rate of 65% in the bone marrow). These 2 approaches should be considered valid options in this setting, although not yet approved. For young fit patients achieving remissions with salvage treatments, the option of allogeneic stem cell transplantation should be discussed as the outcome appears to be unaffected by number and type of previous targeted agents. Novel treatment strategies interfering with different mechanisms of CLL cell survival and proliferation are warranted, including small molecules with novel targets (eg, CDK9, MCL1, ERK inhibitors), CAR T cells targeting different antigens, CAR natural killer cells, or bispecific antibodies.

Kinase inhibitor therapies for Chronic lymphocytic leukaemia (CLL): SYK, BTK and PI3K inhibitors

Chronic lymphocytic leukaemia (CLL) is a prevalent tumor disease in developed countries, and related therapies have been designed. However, CLL is still incurable. Chemoimmunotherapy is effective in inhibiting the proliferation of CLL cells, but nonspecific treatment can affect the growth of other immune cells. Kinase inhibitors are considered to be effective treatments for CLL as their anti-proliferation effects, and currently, popular kinase inhibitor therapies include SYK, BTK, and PI3K inhibitor therapy. PI3K is characterized by high efficiency and low side effects compared with the other two kinase inhibitor therapies, for instance, idelalisib and duvelisib. This review compares the advantages of each kinase inhibitor therapy through relevant studies and concludes that duvelisib has significant advantages and promising prospects compared to other CLL drugs. Further research may focus on exploring the mechanism of the role of kinase inhibitors in CLL as well as the clinical trials of kinase inhibitors in CLL patients.

NRX-0492 degrades wild-type and C481 mutant BTK and demonstrates in vivo activity in CLL patient-derived xenografts

AbstractBruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

High Cell Death Rates at Start of Ibrutinib Therapy Predict for Deeper Remissions in Patients with Chronic Lymphocytic Leukemia (CLL)

Introduction: The BTK inhibitor ibrutinib blocks B-cell receptor and other signaling pathways in CLL cells and has become a standard CLL therapy. Isotopic labeling with deuterated water (2H2O) to measure the effects of ibrutinib on CLL cell proliferation and death directly in patients revealed that ibrutinib profoundly inhibits proliferation and induces high rates of leukemia cell death (registered at clinicaltrials.gov as NCT01752426; Burger et al., JCI Insight, 2017). Here, we report a long-term follow-up of this trial in which we analyzed long-term outcomes and correlated CLL cell birth and death rates with depth of response over time. Methods: Thirty patients with previously untreated CLL/SLL enrolled in the study to receive continuous long-term ibrutinib monotherapy, after one month of labeling with deuterated water. For our analyses, we stratified patients in groups based on high or low CLL cell death rates in the peripheral blood (PB) or lymphatic tissues (lymph nodes, spleen), i.e. death rates above or below the median, assessed over the first months on therapy, as detailed in the original publication. Responses were assessed according to 2008 IWCLL guidelines, with the exception that lymphocytosis was not used as a sole criterion for progression. PB MRD was quantified by flow cytometry with a sensitivity 10-4. Results: The median age of the participants was 64 years (range, 48 - 78); 63.3% were male. Thirteen patients (43%) had advanced stage disease, 57% had unmutated IGHV genes, and 10% carried 17p deletion (del17p). Twenty-two patients (73%) completed 5 years of protocol treatment and subsequently continued treatment with commercial supply ibrutinib. The patients were followed for a median time of 107 months (range, 6 - 111). The reasons for eventual treatment discontinuation (n=21) were adverse events (n=8, 38%), progressive disease (n=4, 19%), change to another treatment (n=5, 24%), death (n=2, 7%) and other reasons (n=2, 7%). To date, nine patients are still receiving ibrutinib. Five patients (17%) achieved a complete remission, 24 (80%) achieved partial remission, and one patient (3%) had stable disease, resulting in an overall response rate of 97%. Median progression-free (PFS) and overall survival (OS) were not reached at a median follow-up of 107 months. Five-year PFS was 80.6%, and OS was 90%. PB MRD was measured in 18 patients after 5 years of ibrutinib (54 to 60 cycles); all patients had detectable MRD (median 11.8%, range, 0.06 - 47.30%). We found that CLL cell death rates in PB and tissues after treatment initiation inversely correlated with the level of PB MRD after 5 years (r=-0.5872, P=0.0104 and r=-0.4407, P=0.0672). MRD levels were significantly lower in patients with high CLL cell death rates in PB (7.03% ± 3.68% vs. 29.46% ± 5.53%, mean ± SEM, P=0.0056) and in tissues (9.73% ± 5.49% vs. 28.89% ± 4.42%, P=0.0155) as compared with patients with lower CLL cell death rates (Figure 1). Tissue CLL cell death rates were significantly higher in patients with IGHV-unmutated CLL compared to mutated (28.88% ± 2.75% vs. 13.16% ± 2.82%, P=0.0005), and MRD levels trended lower (IGHV-unmutated: 10.71% ± 5.39% vs. IGHV-mutated: 28.36% ± 6.02%, P=0.0878). No statistically significant correlation was found between CLL cell birth rate at baseline or after start of ibrutinib and MRD levels after 5 years of treatment. Conclusion: CLL cell death rates during early treatment correlated with achieving lower MRD levels. We found that CLL cell death rates are higher in IGHV-unmutated patients suggesting a higher dependency of IGHV-unmutated clones on BCR signaling and, therefore, resulting in deeper responses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Metabolic Reprogramming of Bone Marrow Stromal Cells in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), tumor cell survival depends on a multitude of supportive signals delivered by the immune and stromal surrounding cells of the microenvironment, follicular reticular cells in the lymph nodes and the bone marrow. Through cell-to-cell direct contact or soluble factors production, the microenvironment promotes chemotaxis, homing, survival and proliferation of CLL cells within the lymph nodes and the bone marrow by activating pathways in the tumor cells. On the other hand, CLL cells are able to educate the surrounding immune and stromal cells to set up a protective environment leading to tumor evasion and production of soluble factors by the laters, which highlights the importance of the crosstalk between the tumor cells and the microenvironment. To gain insight into how CLL cells modify stromal cells, we analyzed the gene expression profile and the proteome of a bone marrow mesenchymal stromal cell line, HS5, as a surrogate of the microenvironment in co-culture with the CLL cell line MEC1. HS5 and MEC1 cells were co-cultured but separated by a transwell to avoid direct contact in order to analyze specifically the effect on the stromal cells of soluble factors produced by CLL cells. After 24 hours of co-culture, both RNA and proteins were extracted from HS5 cells for transcriptome and proteome analyses. Transcriptome analysis using RNA-sequencing identified a total of 99 significantly upregulated and 111 significantly downregulated genes in HS5 cells co-cultured with MEC1 cells compared to HS5 cells alone (>2 fold, adjusted p<0.05). Genes encoding cytokines (IL6, IL1B, IL8, CXCL8), growth factors (CSF2, CSF3), NF-kB signaling members (NFKB2, NFKBIZ, TNFAIP3), adhesion molecules (ICAM1) and glycolysis and oxygen homeostasis regulators (ENO2, CYR61, SOD2, NAMPT) were significantly upregulated. Gene set enrichment analysis (GSEA) of these differentially expressed genes revealed a significant enrichment of the IL6-dependent JAK/STAT3 pathway (Normalized enrichment score NES=2.2), the inflammatory response (NES=2.5), the glycolysis (NES=1.9) and the hypoxia (NES=3.5) (all with False Discovery Rate <0.001). Analysis of the proteomic data revealed less pronounced changes of the differentially expressed proteins but metabolism members appeared among the top upregulated proteins. These proteins included the reductases AKR1B1 and PLOD1, and two mitochondrial proteins involved in the respiratory chain: the acetyl CoA-dehydrogenase MCAD and the subunit of the NADH dehydrogenase NDUFA10. In line with these results, clustering and pathway analysis of the proteomic data showed a protein signature related to the oxidation-reduction process. Altogether, these results suggest that CLL cells produce soluble factors that specifically modify the gene expression profile of stromal cells and induce metabolic reprogramming related to a mitochondrial phenotype. To confirm and strengthen these findings, we assessed the cellular oxygen consumption rate (OCR) and the extra-cellular acidification rate (ECAR) using the Seahorse Assay to quantify the mitochondrial respiration and the glycolysis respectively, in the HS5 cells cultured with conditioned media (CM) issued from primary CLL cells (n=3). Analysis of the OCR showed a significant increase of the maximal respiration (p=0.01) and of the spare respiratory capacity (p=0.03) in HS5 cells with CLL CM as compared to HS5 cells alone, indicating an enhanced mitochondrial respiration. Analysis of the ECAR also revealed a significant increase of the glycolysis capacity (p=0.03) in the HS5 cells upon exposure to CLL CM. In agreement with increased glycolysis, glucose uptake and ATP production were enhanced in HS5 cells cultured with the CM issued from CLL cells. Altogether, these results demonstrate that CLL cells increase the production of energy in surrounding stromal cells by enhancement of the glycolysis and by promoting mitochondrial-based oxidative phosphorylation. This bioenergetic shift induced by CLL cells could enhance stromal cells fitness, which therefore will benefit to tumoral cell survival. We are currently investigating how this crosstalk interferes with the effect of BCR pathway inhibitors or BH3-mimetics. Targeting this axis may represent a therapeutic strategy of interest.

Changes in the T Helper Cell Compartment during Treatment with the BTK Inhibitor Acalabrutinib

Interactions with T helper cells are part of the supportive lymph node microenvironment promoting chronic lymphocytic leukemia (CLL) cell growth and survival. Treatment of CLL patients with ibrutinib, the first-in-class BTK inhibitor, induces profound changes in the T cell compartment, with normalization of elevated T cell numbers and plasma cytokine levels, and diversification of the T cell receptor (TCR) repertoire (Yin Q et al., J Immunol 2017). Acalabrutinib, a second generation BTK inhibitor, is more selective for BTK, with less off-target effects towards other kinases, including IL-2-inducible T cell kinase (ITK), an important kinase for T cell signaling. To characterize acalabrutinib effects on T cell numbers and function in patients with CLL, we analyzed serial samples from 12 acalabrutinib-treated CLL patients at baseline, after 1 week, and after 1, 3, 6, and 12 months of continuous acalabrutinib treatment (with monthly Obinutuzumab added during months 3-9) in an ongoing Phase 2 trial (NCT04505254). Six age-matched healthy donors (HD) were analyzed for comparison. T cell subsets were characterized by flow cytometry. Functional T cell response were assessed after stimulation with phorbol myristate acetate and ionomycin, and proliferation by Ki-67 expression. Prior to treatment, untreated CLL patients had elevated numbers of CD4+ (1864±32/ml) and CD8+ (2533±452/ml, mean T cells which normalized after 12 months of therapy (CD4+ 514±65/ml, p<0.01; and CD8+ 747±271/ml, p<0.05). Two key subsets, T follicular helper cells (Tfh) and T regulatory cells (Tregs), also decreased significantly after 12 months of treatment (Tfh from 2.9±0.4% to 0.2±0.1%, p<0.05; and Tregs from 2.4±0.4% to 0.9±0.2%, p<0.05). Next, we analyzed expression of Tfh and Treg surface markers and found significant down-modulation of CD69 (on Tfh from 20±3.7% to 3.3±0.6%, p<0.001; on Tregs from 10±1.8% to 1.7±0.5%, p<0.01), ICOS (on Tfh from 33±2.6% to 13±1.6%, p<0.01), PD-1 (on Tregs from 7.8±1.3% to 1.2±0.3%, p<0.01), and CTLA-4 (on Tfh from 16±3.2% to 3.8±1.0%, p<0.05; on Tregs from 14±1.5% to 3.1±0.7%, p<0.001) after 12 months of treatment. Additionally, this therapy resulted in reduced cytokine production by Tfh and Tregs when compared to baseline samples, as quantified by flow cytometry after cytoplasmatic staining. For example, induced IL-21 expression decreased from 23±1.3% in baseline samples to 12±0.5% after 12 months, p<0.05 (for comparison, 5.7%±1.3% in HD), IL-10 decreased from 7.3±1.1% to 0.9±0.2% at 12 months, p<0.05 (0.9%±0.1% in HD), and TGFb (from 6.2±0.9% to 0.8±0.2% at 12 months, p<0.01 (1.6%±0.5% in HD). Moreover, circulating Tfh cells from pre-treatment samples expressed higher levels of the co-stimulatory molecule CD40L upon restimulation at baseline compared to after 12 months of treatment and the levels in HD (from 23.0±1.6% to 11.0±1.1% at 12 months, p<0.05; versus 3.8%±1.0% in HD, p<0.001). Looking at Ki-67 expression, we noted, as expected, a significant decrease in CLL cell proliferation from 3.9±0.8% down to 0.8±0.6% after 1 month of therapy (p<0.001), similar data were seen at later time points. Interestingly, Tregs also had reduced Ki-67 expression, down from 16±0.6% to 7.3±0.9% after 6 months (p<0.01) and to 7.9%±1.1% after 12 months of therapy (p<0.05). Collectively, the reduction in T cells and T helper cell subset numbers, along with changes in surface activation markers, and reduced cytokine and Ki-67 expression suggest that an activated pre-treatment T helper cell compartment normalizes during continuous acalabrutinib therapy. These findings are reminiscent of the T cell data from patients receiving ibrutinib therapy, suggesting that off-target effects of the BTK inhibitor may not dictate T helper cell changes, and these may rather be due to activation by the CLL clone, which diminishes as the underlying disease responds to treatment.