B-cell Receptor Signaling Inhibitors Research Articles

Lessons learned from the Eµ-TCL1 mouse model of CLL

The Eµ-TCL1 mouse model has been used for over 20 years to study the pathobiology of chronic lymphocytic leukemia (CLL) and for preclinical testing of novel therapies. A CLL-like disease develops with increasing age in these mice due to a B cell specific overexpression of human TCL1. The reliability of this model to mirror human CLL is controversially discussed, as none of the known driver mutations identified in patients are found in Eµ-TCL1 mice. It has to be acknowledged that this mouse model was key to develop targeted therapies that aim at inhibiting the constitutive B cell receptor (BCR) signaling, a main driver of CLL. Inhibitors of BCR signaling became standard-of-care for a large proportion of patients with CLL as they are highly effective. The Eµ-TCL1 model further advanced our understanding of CLL biology owed to studies that crossed this mouse line with various transgenic mouse models and demonstrated the relevance of CLL-cell intrinsic and -extrinsic drivers of disease. These studies were instrumental in showing the relevance of the tumor microenvironment in the lymphoid tissues for disease progression and immune escape in CLL. It became clear that CLL cells shape and rely on stromal and immune cells, and that immune suppressive mechanisms and T cell exhaustion contribute to CLL progression. Based on this knowledge, new immunotherapy strategies were clinically tested for CLL, but so far with disappointing results. As some of these therapies were effective in the Eµ-TCL1 mouse model, the question arose concerning the translatability of preclinical studies in these mice. The aim of this review is to summarize lessons we have learnt over the last decades by studying CLL-like disease in the Eµ-TCL1 mouse model. The article focuses on pitfalls and limitations of the model, as well as the gained knowledge and potential of using this model for the development of novel treatment strategies to achieve the goal of curing patients with CLL.

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.

T Cell Dysfunction and Exhaustion in Patients with CLL: The Impact of Long Term Ibrutinib Treatment

Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with defects in the immune system and is dependent on the microenvironmental niche for survival. Ibrutinib, the first irreversible inhibitor of Bruton's tyrosine kinase targets B-cell receptor signaling to kill tumor cells. In addition, ibrutinib also appears to regulate the tumor microenvironment and T-cell immunity, but this mechanism unknown. Increased expression of inhibitory receptors, and functional defects such as reduced proliferative capacity and cytotoxicity are common in T cells from patients with CLL. The effect of ibrutinib on T-cell immunity has not been extensively studied in the setting of long term ibrutinib treatment. Therefore, we investigated the phenotype and function of CD4+ and CD8+ T-cells from patients with CLL treated with ibrutinib, treatment naïve CLL and age-matched healthy controls. Methods: CLL patient samples were obtained from treatment naïve patients at diagnosis, and at the 3- and 5-year timepoints. Samples from patients on continuous ibrutinib who had not relapsed at the 3- and 5-year timepoints; and pre-ibrutinib were obtained as well. Age matched healthy donor-derived peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. PBMCs from CLL samples and healthy donors were labeled with CellTrace Violet and treated with anti-CD3 and anti-CD28 (ThermoFisher) for 7 days. T-cells were stimulated to identify proliferation capacity and to identify immune profiles. Results: Efficient T-cell activation and proliferation requires multiple signals such as T-cell receptor (TCR) stimulation, a costimulatory signal, and cytokine production. We analyzed the proliferation of T cells in-vitro upon stimulation with anti-CD3/CD28 beads using CellTrace Violet on PBMCs and found that this stimulation resulted in increased expansion of CD4+ T cells and CD8+ T cells in all the samples. We also found significant difference in the proliferation between untreated and treated CLL samples. Interestingly, proliferating T cells had different expression pattern of the exhaustion markers and inhibitory molecules such as TIM3, TIGIT, LAG3, CD160, KLRG1, CD244 and PD-1 at different phases of the disease. Increased expression of CD160, CD244, and PD1 are the key features of T-cell exhaustion. We found significant increase in the percentage of T cells from CLL patients expressing CD244, CD160 and PD1 compared to healthy donor controls but not between naïve and ibrutinib treated samples. Long term ibrutinib treatment in 3yr and 5yr samples does not change the exhaustion state in patients. We also found T-cells from ibrutinib treated samples express higher levels of CD57 which is known as a marker of senescence (Figure 1A). The phenotypes of untreated CLL samples are CCR7+CD45RA- central memory T-cells (35%) and CCR7+CD45RA+ naïve T cells (25%) (p<0.05) after stimulation in-vitro on day 3. In the same setting, ibrutinib treated samples had higher levels of effector memory T-cells (p<0.05) (80%) compared to untreated CLL samples (Figure 1B). These results suggest that the phenotype has changed to central memory phenotype in untreated samples. Functional T-cell exhaustion in CLL also leads to failure in cytokine production. Therefore, we measured different cytokine levels after stimulation in all the patient samples and in healthy controls. We found significant increase in the levels of inflammatory cytokines such as IFN-γ (p<0.05) and TNF-α (p<0.05) in long term ibrutinib treated samples compared to naïve CLL samples and healthy controls. Conclusion: Several studies have showed that T-cell counts are increased after treatment with ibrutinib in patients with CLL but not in extended therapy. Our findings suggest that long term ibrutinib treatment does increase T-cell number and proliferative capacity in CLL. The proliferating T cells express higher levels of exhaustion markers and inhibitory molecules in-vitro making them dysfunctional. However, these T-cells still maintained good proliferative potential, on par to healthy donors and better than untreated patients which can be applied in expanding improved T cell grafts for various types of adoptive T cell therapy, such as TCR gene therapy, and CAR therapy. Also better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells.

Role of Autoantigen Induced-B-Cell Receptor Internalization and Its Inhibition for the Biology and Survival of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

In normal B-cells, antigen binding by the B cell receptor (BCR) triggers rapid BCR clustering, initialization of signaling, and subsequent endocytosis of the BCR-antigen complex. Numerous studies showed that BCR signaling is continued as the BCR is trafficked intracellularly. Similarly, the internalized BCR in complex with TLR9 in DLBCL cells elicits crucial pro-survival signaling for DLBCL cells. Despite these observations, the role of endocytosis in generating a pathological signal in DLBCL, and a potential of endocytosis blockade as a strategy to inhibit BCR signaling, have not been defined. To address these questions, we generated DLBCL cell models with modified, ovoalbumin (OVA)-specific hypervariable regions (HVR) in BCR using the knock-in CRISPR-Cas9 methodology. This model allows to study BCR receptor internalization and the signaling that accompanies this process in highly controlled conditions. The change of BCR specificity led to inhibition of spontaneous (autoantigen-initiated) BCR internalization, observed as an increased surface BCR level, and lower proliferative potential of autoautigen-dependent U2932 and HBL1 cells, but not in cells in which autoantigens were not detected (LY19). Using the kinome analysis platform (PamGene technology), we identified kinases activated by BCR-autoantigen binding, including proximal mediators of BCR signaling, cyclin-dependent kinases, protein kinase C and S6 ribosomal kinases. Simultaneous analysis of the OVA-induced BCR internalization kinetics and signaling activation in the above-described models indicated that the activation of certain signaling cascades may emanate from the inside of the cell. Using the Proximity Ligation Assay (PLA) method, we identified the presence of BCR-TLR9-IkB complexes (activators of the NF-kB transcription factor), in the endosomal compartment of autoantigen-dependent DLBCL cells. Cells with OVA-specific BCR showed a drastically decreased number of BCR-TLR9-IkB complexes, which was associated with lower expression of NF-kB-dependent genes. To confirm the crucial role of the autoantigen-induced BCR endocytosis in sustaining the signaling and promoting DLBCL cells survival, we reanalyzed available data from the CRISPR-Cas9-Screen studies. This approach revealed the key role of genes involved in endocytosis, including dynamin-2, for DLBCL cell survival. Based on these observations, we generated cell models characterized by inducible expression of the dominant-negative (DN) form of dynamin-2, which inhibits dynamin-2-dependent endocytosis. Experiments performed using these models showed that BCR internalization is dependent on dynamin-2, and the induction of the DN-dynamin-2 form blocks internalization of the BCR receptor, BCR-TLR9-IkB complexes assembly and activation of BCR-initiated signaling pathways, including the transcription factor NF-kB, which markedly inhibited the growth of DLBCL cells. In addition, clinically available Dynamin-2 inhibitors (e.g. certain phenothiazine derivatives), also inhibited BCR endocytosis and were toxic to DLBCL cells. In summary, our observations demonstrate that autoantigen-induced endocytosis of the BCR receptor is the key mechanism supporting the BCR signal and ensuring the survival of DLBCL cells with autoantigen-specific BCRs. Furthermore, we show that endocytosis is a rational therapeutic target in this group of lymphoid malignancies.

The PKC-β Inhibitor MS-553 Displays Preclinical Efficacy in BTK Inhibitor Resistant Chronic Lymphocytic Leukemia

Background: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by usage of targeted therapies against the B-cell receptor (BCR) signaling cascade, specifically Bruton's Tyrosine Kinase (BTK). However, resistance to BTK inhibition occurs through acquisition of various mutations in BTK or its immediate downstream signaling partner, PLCγ2. Clinical outcomes for these patients are poor, emphasizing the need for alternative therapeutics. In the presence of these mutations, BCR signaling remains intact, suggesting that targeting molecules downstream of BTK may be effective. Protein kinase C-β (PKCβ) is a downstream component of the BCR pathway that has been shown to be over-expressed in CLL. Moreover, PKCβ is essential to the development of CLL in the Eμ-TCL1 mouse model and its expression in stromal cells is required for the survival of leukemic B-cells (Holler et al. 2009). This suggests that signaling through PKCβ, in both CLL cells and cells in the microenvironment, is essential to promote leukemogenesis. MS-553 is a potent, ATP competitive, reversible inhibitor of multiple PKC isoforms including PKCβ, with the potential to overcome BTKi mediated resistance. Methods: Primary CLL cells were isolated by negative selection and treated with up to 5 μM MS-553. Cell viability was assessed via Annexin V/PI with and without HS-5 stromal cell co-culture. BCR and canonical WNT signaling changes were evaluated by change in target protein phosphorylation or total protein by immunoblot following drugging. Changes in downstream NFκB and WNT signaling were interrogated by RT-qPCR. Using genetically modified TMD8 cells that express only C481S or T474I mutated BTK, we tested MS-553's effect on cell-viability and BCR signaling. In vivo survival studies were performed using the Eμ-MTCP1 (Walker et al. 2021) adoptive transfer mouse model, an aggressive model of CLL, with subsequent randomization into vehicle or MS-553 treatment arms. Results: Following 72 hours of drugging, 5 μM MS-553 displayed modest cytotoxicity against primary CLL cells both alone (50%, p<0.0001, n=8) and in the presence of HS-5 stromal cells (25%, p=0.0008, n=8). In primary CLL cells harboring the C481S BTK mutation, we observed retained cytotoxicity following 72 hours of drugging (29%, p=0.00029, n=6). At 24 hours, 5 μM MS-553 inhibited downstream BCR and WNT signaling in primary CLL cells, demonstrated by reduced phosphorylation of PKC-β (43%, p=0.0024, n=15) and several of its downstream targets including pGSK3β (45%, p<0.0001, n=15), pIκBα (66%, p<0.0001, n=15), and pERK (17%, p=0.0301, n=15), and reduced total protein of β-Catenin (82%, p<0.0001, n=9), Cyclin D2 (71%, p=0.0011, n=9), and cMyc (85%, p<0.0001, n=9) compared to vehicle treated, stimulated samples. In both treatment naïve and C481S patient samples, mRNA expression of NFκB and WNT pathway targets BCL-2, OCT-2, BFL-1, MYC, CD40, Cyclin D1, and Cyclin D2 were all decreased after 48 hours of treatment with 1 or 5 μM MS-553. MS-553 also showed efficacy in the presence of BTK mutations: viability in TMD8 cells was reduced following 72-hour drugging with MS-553 regardless of BTK mutational status (WT: 22%, p=0.0006, n=3; C481S BTK: 18%, p=0.0032, n=3; T474I BTK: 19%, p=0.0015, n=3). Inflammatory cytokine production is inhibited by MS-553 through its ability to decrease CCL3 and CCL4 cytokine expression in WT and mutant BTK-harboring TMD8 cells (p<0.0001, n=3) (Figure 1A). To validate inhibition of BCR signaling in the C481S BTK setting, we treated primary CLL cells harboring this mutation and observed a reduction in both CCL3 and CCL4 production (61%, p=0.001229, n=5). As proof of concept, we examined MS-553's efficacy in vivo in the Eμ-MTCP1 mouse engraftment model, and found a significant reduction in disease progression and increase in survival (median 117 days) when compared to vehicle control (median 56 days, p=0.0003) (Figure 1B). Conclusion: Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL including that with BTK mutations conferring BTKi resistance. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al. 2022).

MS-553, a Selective PKC-ß Inhibitor Inhibits BCR Signaling and Synergizes with Venetoclax for the Treatment of High Risk CLL

Inhibitors of Bruton's tyrosine kinase (BTK) have been transformative in treating patients with chronic lymphocytic leukemia (CLL) due to their ability to inhibit B-Cell Receptor (BCR) signaling. However, despite durable remissions in many patients, complete responses are uncommon and acquired mutations in BTK and its downstream target phospholipase Cγ2 (PLCγ2) lead to relapses and poor survival. In parallel, genomic high-risk features such as aberrations in TP53 represent an independent risk factor that favor progression on BTK inhibitors (BTKi). TP53 aberrations comprise of deletions in chromosome 17 (del17p) which result in the loss of one allele of TP53 and point mutations in the TP53 gene. CLL patients with del17p acquire clonal mutations that affect the other p53 allele in 82% of cases and largely express only mutant TP53 protein. Therefore, there is an urgent need to identify therapies for patients who progress on BTKi. We have observed over-expression of protein kinase C-β (PKC-β) in primary CLL patient samples including in those that have mutations in TP53. PKC-β is a downstream of the BCR and functions downstream of BTK. PKC-β is required for CLL cell survival and proliferation and therefore can be a potential target for patients with mutations in p53 that do not respond to other targeted therapies. We hypothesized that targeting PKC-β will inhibit BCR signaling downstream of BTK and may offer an effective therapeutic strategy for patients who relapse on ibrutinib. Mechanistically, we found that CLL cells (n=6 patients) in contact with stromal support upregulated their levels of PKC-β. Pre-exposure of stromal cells to MS-553, a selective PKC-β inhibitor prevented the stromal cell contact-induced increase in PKC-β within the CLL cells establishing that stromal contact was an important source of PKC-β in CLL cells. We have shown in primary CLL patient samples that exposure to MS-553 decreases activation of PKC-β and its downstream effectors GSK3-β and ERK in a dose-dependent manner regardless of mutations in p53 both in the presence and absence of stromal support. In parallel, MS-553 also decreased the levels of BCL-xL, a key pro-survival BCL2 family protein via downregulation of β-catenin. Using dynamic BH3 profiling we show that CLL samples (both WT and mutant TP53) exposed to MS-553 (5µM, 24h) increase their dependence on BCL-2 and BCL-xL for survival. This finding suggested that MS-553 could synergize with the BCL-2 inhibitor venetoclax to eliminate CLL cells. Correspondingly, cells exposed to a combination of MS-553 and venetoclax released more cytochrome c (Cyt c), used as a measure of mitochondrial dysfunction, compared to each single agent alone and led to a synergistic increase in apoptosis as measured by Annexin V positivity. These data provide strong rationale for an ongoing Phase I/II trial that evaluates MS-553 as a single agent and in combination with targeted agents such as venetoclax. Early interim data indicate that MS-553 is safe, tolerable and effective both as a single agent and in combination with venetoclax.

Genomic Characterization of Patients in a Phase 2 Study of Zanubrutinib in BTK Inhibitor-Intolerant Patients with Relapsed/Refractory B-Cell Malignancies

Background/Introduction: Targeting Bruton tyrosine kinase (BTK) to inhibit B-cell receptor signaling is an effective way to treat B-cell malignancies. However, some patients (pts) have experienced toxicities to BTK inhibitors ibrutinib (ibr) and acalabrutinib (acala), which lead to dose reduction or treatment discontinuation. Zanubrutinib (zanu) is a potent and selective next-generation BTK inhibitor (Blood. 2021;138[suppl 1]:1410). BGB-3111-215 (NCT04116437) is an ongoing, phase 2 study of the safety and efficacy of zanu monotherapy in pts with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Waldenström macroglobulinemia (WM), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL) who discontinued ibr, acala, or acala and ibr because of intolerance. Here, we present the gene mutation profile of pts in this study to demonstrate the mutational landscape of pts intolerant to ibr or acala and to explore the association between gene mutations and response to zanu in ibr- and acala- intolerant pts. Methods: Peripheral blood from pts was collected before treatment and at or after the time of disease progression. Genomic DNA was isolated from peripheral blood mononuclear cells or plasma, and the mutational status of targeted genes was assessed using a targeted 106-gene next-generation sequencing (NGS) panel (PredicineHEMETM, Predicine). Samples were sequenced to a median depth of >20,000 reads, with a validated sensitivity of 0.25% mutant allele frequency for all genomic regions. Results: Samples from 63 pts were analyzed, including 63 at baseline and 5 at or after progressive disease (PD). There were 41 pts with CLL, 6 with SLL, 10 with WM, 3 with MCL, and 3 with MZL. The most common mutations at baseline were TP53 (31.7%), SF3B1 (22.2%), ATM (17.5%), NOTCH1 (17.5%), CHEK2 (14.3%), and KRAS (12.7%). Copy number variants were detected, including deletions at ATM locus (9.5%) and RB1 locus (9.5%) and amplifications at CCND2 locus (6.3%). For CLL/SLL, the frequencies of TP53 (27.7%), NOTCH1 (21.3%), SF3B1 (25.5%), ATM (21.3%), and BIRC3 (10.4%) mutations were comparable to those in other studies with high-risk relapsed/refractory CLL pts (Leukemia. 2018;32:3-91; Blood. 2014;123:3247-3254; Leuk Lymphoma. 2018;59[10]:2318-26). For WM, 5 of 10 (50%) samples harbored TP53 mutations, and 4 of 10 samples had MYD88 mutations; all harbored the L265P mutation. Among the 4 pts with MYD88 mutation, CXCR4 gene mutation was detected in 1 case. All 6 pts with MYD88 wild type also had CXCR4 wild type gene, consistent with published data (N Engl J Med. 2015;373:584-6). Baseline mutations were available for the 10 pts with PD, including 8 CLL, 1 SLL, and 1 MCL. Compared to pts without PD, pts with PD had increased frequency of certain mutations: TP53 (60%, P=0.071), ATM (50%, P=0.017), SF3B1 (50%, P=0.035), RB1 (40%, P=0.009), SETD2 (40%, P=0.009), and CDKN2A (30%, P=0.011). This finding suggests that these particular baseline mutations are associated with resistance to zanu. Five pts also had mutations detected at or after PD. Of these, 3 pts had BTK or PLCG2 mutations (submitted). One patient with CLL and without BTK or PLCG2 mutations had ATM and FBXW7 mutations (variant allele frequencies were 86% and 50%, respectively), suggesting DNA damage pathway mutations and NOTCH1 dysregulations may have contributed to disease progression in this patient. Conclusion: Exploratory analysis results confirmed that cell cycle, DNA damage, and NOTCH1 pathway genes were frequently mutated in pts with B-cell malignancies on study BGB-3111-215 (pts intolerant to ibr and/or acala). Pts with mutations associated with poor prognosis at baseline were more likely to develop PD.

B-cell Receptor Signaling Induced Metabolic Alterations in Chronic Lymphocytic Leukemia Can Be Partially Bypassed by TP53 Abnormalities.

It has been unclear what role metabolism is playing in the pathophysiology of chronic lymphocytic leukemia (CLL). One reason is that the study of CLL metabolism is challenging due to the resting nature of circulating CLL cells. Also, it is not clear if any of the genomic aberrations observed in this disease have any impact on metabolism. Here, we demonstrate that CLL cells in proliferation centers exhibit upregulation of several molecules involved in glycolysis and mitochondrial metabolism. Comparison of CXCR4/CD5 intraclonal cell subpopulations showed that these changes are paralleled by increases in the metabolic activity of the CXCR4lowCD5high fraction that have recently egressed from the lymph nodes. Notably, anti-IgM stimulation of CLL cells recapitulates many of these metabolic alterations, including increased glucose uptake, increased lactate production, induction of glycolytic enzymes, and increased respiratory reserve. Treatment of CLL cells with inhibitors of B-cell receptor (BCR) signaling blocked these anti-IgM-induced changes in vitro, which was mirrored by decreases in hexokinase 2 expression in CLL cells from ibrutinib-treated patients in vivo. Interestingly, several samples from patients with 17p-deletion manifested increased spontaneous aerobic glycolysis in the unstimulated state suggestive of a BCR-independent metabolic phenotype. We conclude that the proliferative fraction of CLL cells found in lymphoid tissues or the peripheral blood of CLL patients exhibit increased metabolic activity when compared with the bulk CLL-cell population. Although this is due to microenvironmental stimulatory signals such as BCR-engagement in most cases, increases in resting metabolic activity can be observed in cases with 17p-deletion.

Modeling the B-cell receptor signaling on single cell level reveals a stable network circuit topology between nonmalignant B cells and chronic lymphocytic leukemia cells and between untreated cells and cells treated with kinase inhibitors.

B-cell receptor (BCR) signaling is central for the pathomechanism of chronic lymphocytic leukemia (CLL), and inhibitors of BCR signaling have substantially improved treatment options. To model malignant and nonmalignant BCR signaling, we quantified five components of BCR signaling (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) in single cells from primary human leukemic cells and from nonmalignant tissue. We measured signaling activity in a time-resolved manner after stimulation with BCR crosslinking by anti-IgM and/or anti-CD19 and with or without inhibition of phosphatases with H2 O2 . The phosphorylation of BCR signaling components was increased in malignant cells compared to nonmalignant cells and in IGHV unmutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2 O2 led to higher phosphorylation levels of BCR components in CLL cells with mutated IGHV compared to unmutated IGHV. We modeled the connectivity of the cascade components by correlating signal intensities across single cells. The network topology remained stable between malignant and nonmalignant cells. To additionally test for the impact of therapeutic compounds on the network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib), LYN (dasatinib) and SYK (entospletinib). Idelalisib treatment resulted in similar effects in malignant and nonmalignant cells, whereas ibrutinib was mostly active on CLL cells. Idelalisib and ibrutinib had complementary effects on the BCR signaling cascade whose activity was further reduced upon dasatinib and entospletinib treatment. The characterization of the molecular circuitry of leukemic BCR signaling will allow a more refined targeting of this Achilles heel.

Abstract P073: Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors

Abstract Background. Marginal zone lymphoma (MZL) is a heterogeneous B-cell malignancy for which no standard treatment exists. B-cell receptor (BCR) signaling dysregulation is a hallmark of MZL and can mimic antigen dependent BCR activation. However, single treatment with BCR signaling inhibitors shows limited complete response rate and relapsing patients. IRAK4 is a protein kinase downstream the Toll-like receptor signaling. CA-4948 is the first-in-class inhibitor of IRAK4 and it is in phase 1/2 studies for patients with relapsed/refractory clinical studies with favorable activity in lymphomas and myeloid neoplasms. Here, we assessed IRAK4 inhibition with CA-4948 against a panel of MZL cell lines in which we have developed secondary resistance to inhibitors of PI3K (idelalisib, copanlisib) or BTK (ibrutinib). Methods. Models with secondary resistance were developed by continuous exposing cell lines derived from splenic MZL (Karpas 1718 and VL51) to high doses of drugs for long period of times. Resistant and parental lines were exposed to increasing doses of CA-4948, alone or in combination with compounds they are resistant to. Cell viability was tested by MTT assay (72h treatment). Synergy of combinations was evaluated according to the Chou-Talalay combination index (CI), as well as potency and efficacy, estimated according to the MuSyC algorithm. Results. The parental Karpas1718, bearing a MYD88 L265P mutation, had an IC50 of 3.29 µM for CA-4948 as single agent, while the parental VL51 and all resistant models presented IC50 values in the range of 19-35 µM. IRAK4 inhibition with CA-4948 had strong benefit when combined to downstream BCR inhibitors. In resistant cells, CA-4948 was strongly synergistic with idelalisib, ibrutinib and, at lesser extent, with copanlisib. CA-4948 in combination with ibrutinib was strongly synergistic especially in the VL51 ibrutinib resistant model compared to the parental one. CA-4948 (from 1 to 5 µM) recovered sensitivity to ibrutinib at IC50 values close to the parental condition. Similarly, CA-4948 in combination with idelalisib was also synergistic and increased sensitivity to idelalisib in idelalisib resistant VL51. Less benefit was seen adding CA-4948 to copanlisib: the combination was synergistic in VL51 parental cell, but with only weak efficacy. In the Karpas1718, CA-4948 was synergistic either in idelalisib resistant or in parental, with improved benefit in the latter, in which synergistic effect was observed when combined with both idelalisib and ibrutinib. The synergistic activity of CA-4948 was mainly due to improved efficacy rather than potency of the combinatorial partners. Conclusions. Our results in MZL cell lines show that the IRAK4 inhibitor CA-4948 is synergistic with small molecules targeting BCR signaling, especially with idelalisib and ibrutinib. Data also suggest that concentrations of CA-4948 comparable to those achieved in patients might overcome or reduce secondary resistance to the tested tyrosine kinase inhibitors. Citation Format: Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Laura Barnabei, Reinhard von Roemeling, Elizabeth Martinez, Emanuele Zucca, Francesco Bertoni. Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P073.

Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome.Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it.To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance.The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment.To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells.Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002).Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. DisclosuresBiondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Case Presentation – Relapse After Frontline BTKi Therapy in Patients with CLL: Options and Consideration

AbstractThe advent of small molecule inhibitors of B-cell receptor (BCRi) signaling has revolutionized the therapy for patients with chronic lymphocytic leukemia (CLL). These therapies have largely supplanted cytotoxic chemotherapy for both untreated and relapsed disease.1,2 The Bruton’s tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib, have demonstrated high rates of durable responses and prolonged progression free survival (PFS) and are approved for the treatment of previously untreated or relapsed CLL.3–11 Zanubrutinib, which is already approved for mantle cell lymphoma,12 is likely to gain approval for CLL in the near future, and there are other BTKi inhibitors including noncovalent BTKis that are currently in development.

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

RAC2 Links B-Cell Receptor Signaling and Cell Adhesion in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin B-cell lymphoma. B-cell receptor (BCR) signaling is chronically active in several mature B-cell malignancies including mantle cell lymphoma. Inhibition of the BCR and downstream pathway is highly effective in B-cell neoplasia and can be achieved at the cellular level through BTK inhibition by ibrutinib, for example. Ibrutinib has generated remarkable patients' response in large clinical trials and has been approved by FDA for the treatment of relapsed/refractory MCL. Ibrutinib targets Bruton tyrosine kinase primarily by inhibition of B-cell receptor signaling and cell proliferation, and secondarily by induction of tumor apoptosis. In addition, the drug inhibits the interaction of tumor cells with their microenvironment by disruption of cell adhesion. The effect on cell adhesion appears to be a mechanism shared by many BCR-targeted therapies including inhibitors of LYN (Leuk Res 38, 34, 2014), SYK (Blood, 115, 2578, 2010; Blood 125, 2336, 2015), and PI3K (idelalisib) (NEJM, 370, 997, 2014; Blood, 125, 2306). This class action raised a question regarding how the BCR pathway is connected to the cell adhesion phenotype. We set out to investigate the underlying mechanisms that are largely unknown at the present time.Methods: We used RNA-sequencing to identify pathways that have differential responses to ibrutinib in MCL cell lines that are either intrinsically sensitive or resistant to ibrutinib. We then validate the RNA-Seq findings in sensitive and resistant cell lines via RNA interference and transfection, xenograft mouse models, and human primary MCL samples.Results: We found that sensitivities of six MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. These include sensitive cell line Jeko-1, intermediate cell lines Mino, REC-1 and JVM2, and resistant lines Maver-1 and Granta 519. RNA-sequencing revealed that BCR signaling and cell adhesion transcriptional programs were coordinately down-regulated by ibrutinib in ibrutinib-sensitive cells, but not in ibrutinib-resistant cells. Among genes that are differentially regulated in the sensitive and resistant cell lines, we identified RAC2, a regulator of cell adhesion this is also involved in BCR signaling. To determine whether RAC2 is relevant, we examined RAC2 expression in primary MCL tumors by immunohistochemical staining (IHC). We found that RAC2 was weakly expressed in 7/10 classic MCL tumors and strongly expressed in 3/3 aggressive MCL. In contrast, in tonsilar lymphoid reactive tissues, RAC2 positivity was restricted only to mantle zone and T-zone of the follicles. For further validation, we found that RAC2 expression, at the protein level, was down-regulated by ibrutinib treatment in sensitive, but not resistant, cell lines. More interestingly, using co-immunoprecipitation, we found that RAC2 is physically associated with BLNK, an adaptor of early BCR signaling pathway, in the sensitive cells but not in the resistant cells. To establish that RAC2 is a regulator of MCL cell adhesion in the context of MCL, we demonstrated that siRNA knock-down of RAC2 impaired cell adhesion while exogenous introduction of RAC2 reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib showed slower tumor growth with reduced RAC2 expression by IHC. Lastly, we show that in primary human MCL cells, RAC2 was down-regulated by ibrutinib, and this down-regulation correlated well with cell adhesion impairment following ibrutinib treatment.Conclusions: Our findings suggest the RAC2 mediates the link between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC expression and via disruption of RAC2-BLNK interaction. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development. DisclosuresLu:Portola Pharmacueticals, Inc.: Research Funding. Wang:Portola: Research Funding; Karyopharm: Research Funding.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled By Functional Lipoprotein Nanoparticles and the B Cell Receptor Inhibitor Ibrutinib: A Therapeutic Strategy Dependent on Cell of Origin (COO)

Introduction: Malignant cells, including lymphoma cells, have altered metabolism and, in some cases, an increased demand for cholesterol and cholesteryl esters. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative, exciting approach. Toward this end, we focused on DLBCL as a model because of differences in cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed bio-inspired high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol and cholesteryl ester uptake through binding to the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). We hypothesized that by inhibiting BCR signaling, and consequently cholesterol biosynthesis with the BTK inhibitor ibrutinib, we could overcome inherent resistance in cholesterol replete ABC DLBCL cell lines in vitro and in a xenograft model.Methods: HDL NPs were synthesized by surface-functionalizing a gold nanoparticle core as previously described (Thaxton et al JACS 2009) with the HDL defining apolipoprotein A-I and phospholipids, with the resultant HDL NPs having similar size, surface composition, charge and function as natural HDLs. However, owing to the gold nanoparticle core, HDL NPs are not an appreciable source of cholesterol or cholesteryl esters. GC DLBCL (SUDHL4) and ABC DBLCL (TMD8, HBL-1) cell lines were treated with HDL NPs as monotherapy or in combination with BCR signaling inhibitors (e.g. ibrutinib), and assayed for total cholesterol content and cell viability. Quantitative PCR was conducted to measure changes in expression of cholesterol biosynthesis genes. TMD8 tumor xenografts were initiated in SCID-beige mice, and following implantation, the mice were treated with vehicle, HDL NPs, ibrutinib, or a combination of the two treatments.Results: As we have previously reported (Yang et al PNAS 2013), GC DLBCL are exquisitely sensitive to HDL NP as monotherapy while ABC DLBCL are less sensitive. Here, our data demonstrate that HDL NPs, as a monotherapy, statistically significantly reduced total cellular cholesterol levels in SUDHL4 cells (7.96 ± 1.47 µg cholesterol/ 104 cells for 0nM HDL NP vs. 2.07 ± 2.07 µg cholesterol/ 104 cells for 100nM HDL NP; p=0.0007), resulting in profound cell death (HDL NP IC50 = 2nM). Further, HDL NP-induced cholesterol depletion resulted in upregulation of numerous cholesterol biosynthesis genes in SUDHL4 cells, including HMG-CoA synthase 1, 7-dehydrocholesterol reductase, lanosterol synthase, and others. In ABC DLBCL cells, the enhanced BCR signaling and resultant de novo cholesterol synthesis drastically diminishes the ability of HDL NPs to reduce cellular cholesterol (TMD8: 12.80 ± 5.08 µg cholesterol/ 104 cells for 0nM HDL NP vs. 16.35 ± 1.17 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.84; HBL-1: 29.16 ± 5.96 µg cholesterol/ 104 cells for 0nM HDL NP vs. 23.51 ± 5.65 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.75) and induce cell death (TMD8 HDL NP IC50 = 69nM; HBL-1 HDL NP IC50 = 75nM). Additionally, HDL NPs had little to no effect on the expression of the cholesterol biosynthesis genes in the ABC cell lines. To reduce cholesterol biosynthesis, which depends on BCR signaling, we combined HDL NPs with the BCR signaling inhibitor ibrutinib. We found that HDL NP synergize with ibrutinib in the ABC derived TMD8 cell line in vitro and in a xenograft model. Correspondingly, HDL NPs and ibrutinib reduced the total cellular cholesterol content of TMD8 and HBL-1 cells when administered together, but had no effect when given alone. Thus, by targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis we achieved synergistic cellular cholesterol reduction and subsequent cell death, both in vitro and in the TMD8 in vivo tumor xenograft model.Conclusion: These results in DLBCL lymphoma cell lines and tumor xenografts demonstrate that depletion of cellular cholesterol, in a specifically targeted manner dependent on the HDL receptor SCARB1 and differential BCR signaling in ABC vs GCB cells, is a powerful mechanism to induce apoptosis in lymphoma and opens a new paradigm in lymphoma biology and therapy. DisclosuresThaxton:Aurasense: Other: The HDL NPs technology was licensed by Northwestern University to a biotech company (Aurasense) founded by C. Shad Thaxton . Gordon:Janssen: Other: Data Monitoring Committee.

External Stimuli Modulate EZH2 Expression in Chronic Lymphocytic Leukemia. Rationale and Evidence for Synergistic Anti-Tumor Effects of B Cell Signaling and EZH2 Inhibitors

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). We have previously reported that EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Moreover, we demonstrated that EZH2 pharmacological inhibition induced down-regulation of H3K27me3 levels leading to increased cell apoptosis. Here we sought to investigate if EZH2 expression could be affected by microenvironmental triggering, a key driver of CLL development and evolution.The study group included 10 CLL cases (9 with unmutated and 1 with mutated IGHV genes) that had previously been screened and found responsive to cross-linking of the B cell receptor (BcR) with anti-IgM. CD19+ cells were negatively selected from peripheral blood and stimulated or not through the BcR for 14 hours. Using RQ-PCR we found that stimulation with anti-IgM showed opposing effects on EZH2 mRNA levels, since 5 cases upregulated while the remaining 5 downregulated EZH2. In contrast, stimulation with CpG or CpG/CD40L for 14 hours caused pronounced and consistent upregulation of EZH2 mRNA levels compared to unstimulated cells (FD=8.7, p=0.03 and FD=11.3, p=0.03, respectively; n=6). This effect was also obvious at the protein levels in cells treated with CpG, CD40L and CpG/CD40L for 24 hours, where EZH2 protein was significantly upregulated as revealed by Western blot analysis (FD=3.9, p=0.002; FD=1.7, p=0.005; and FD=2.9, p=0.01, respectively; n=6). In CpG-stimulated cultures, the induction of EZH2 was accompanied by a significant increase in cell viability (FD=2, p=0.002; n=6), supporting the conclusion that EZH2 expression likely contributes to the anti-apoptosis phenotype induced in CLL cells by microenvironmental signals. Prompted by these observations, we then investigated whether B cell signaling inhibition might also modulate EZH2 expression and H3K27me3 levels in vivo by longitudinal profiling of another group of 9 CLL patients that were under ibrutinib treatment. In patients at +1 month under ibrutinib, when, as shown by us and others, CLL cells become anergized through the BcR, we observed a significant decrease in EZH2 protein (FD=3, p=0.0007; n=9) as well a trend for reduced H3K27me3 levels (FD=1.4, p=0.07; n=6) compared to the pre-treatment samples, further highlighting the links between BcR signaling and EZH2 expression and functionality. Next, we assessed ex vivo the effects of ibrutinib on CLL cell viability in cases with differential EZH2 expression [EZH2high (n=7) vs EZH2low (n=6)] and found decreased viability in treated cells compared to control cells (DMSO-exposed) in EZH2high cases (FD=1.9, p=0.0025), with no difference in EZH2low cases.Finally, we examined potential synergistic effects of B cell signaling inhibitors [ibrutinib (IB), idelalisib (IDE)] and EZH2 inhibitors (GSK343, GSK126) in primary CLL cells. To this end, CD19+ cells from 6 CLL cases were first pre-stimulated with CpG for 24 hours and then exposed to either single inhibitors (IB, IDE, GSK343, GSK126) or combinations of B cell signaling and EZH2 inhibitors (IB/GSK343, IDE/GSK343, IB/GSK126). Flow cytometry analysis after 3 days in culture revealed significantly lower CLL cell viability and H3K27me3 levels in CLL cells exposed to combined versus single treatment (FD≥ 1.5, p<0.05).In conclusion, we show that EZH2 expression is affected by signals emanating from CLL microenvironment. Moreover, we provide for the first time evidence that the combination of BcR signaling inhibitors widely used in CLL therapy and EZH2 inhibitors currently tested in phase I/II clinical trials in lymphoid malignancies has synergistic effects, suggesting that targeting both processes might represent a rational and promising therapeutic strategy. DisclosuresStavroyianni:Gilead Sciences: Honoraria; Takeda: Honoraria; Janssen Pharmaceuticals: Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Papakonstantinou:Gilead: Research Funding; Janssen Pharmaceuticals: Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding.

A Deep Insight Into CAR-T Cell Therapy in Non-Hodgkin Lymphoma: Application, Opportunities, and Future Directions.

Non-Hodgkin’s lymphoma (NHL) is a cancer that starts in the lymphatic system. In NHL, the important part of the immune system, a type of white blood cells called lymphocytes become cancerous. NHL subtypes include marginal zone lymphoma, small lymphocytic lymphoma, follicular lymphoma (FL), and lymphoplasmacytic lymphoma. The disease can emerge in either aggressive or indolent form. 5-year survival duration after diagnosis is poor among patients with aggressive/relapsing form of NHL. Therefore, it is necessary to understand the molecular mechanisms of pathogenesis involved in NHL establishment and progression. In the next step, we can develop innovative therapies for NHL based on our knowledge in signaling pathways, surface antigens, and tumor milieu of NHL. In the recent few decades, several treatment solutions of NHL mainly based on targeted/directed therapies have been evaluated. These approaches include B-cell receptor (BCR) signaling inhibitors, immunomodulatory agents, monoclonal antibodies (mAbs), epigenetic modulators, Bcl-2 inhibitors, checkpoint inhibitors, and T-cell therapy. In recent years, methods based on T cell immunotherapy have been considered as a novel promising anti-cancer strategy in the treatment of various types of cancers, and particularly in blood cancers. These methods could significantly increase the capacity of the immune system to induce durable anti-cancer responses in patients with chemotherapy-resistant lymphoma. One of the promising therapy methods involved in the triumph of immunotherapy is the chimeric antigen receptor (CAR) T cells with dramatically improved killing activity against tumor cells. The CAR-T cell-based anti-cancer therapy targeting a pan–B-cell marker, CD19 is recently approved by the US Food and Drug Administration (FDA) for the treatment of chemotherapy-resistant B-cell NHL. In this review, we will discuss the structure, molecular mechanisms, results of clinical trials, and the toxicity of CAR-T cell-based therapies. Also, we will criticize the clinical aspects, the treatment considerations, and the challenges and possible drawbacks of the application of CAR-T cells in the treatment of NHL.

POS0006 NX-5948, A SELECTIVE DEGRADER OF BTK, SIGNIFICANTLY REDUCES INFLAMMATION IN A MODEL OF AUTOIMMUNE DISEASE

Background:Aberrant activation of B cells and autoantibody mediated tissue damage are hallmarks of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Therefore, novel treatments that prevent autoantibody generation or antibody-mediated end organ tissue damage are of high interest. Bruton’s tyrosine kinase (BTK) transduces signals downstream of the B cell receptor (BCR), toll-like receptors, and Fc receptors in B cells and myeloid cells [1]. Overexpression of BTK in B cells leads to hyperactive BCR signaling, plasma cell generation, autoantibody secretion, and an SLE-like disease in mice [2]. Conversely, reducing BTK expression in B cells can ameliorate disease in Lyn-deficient mice.[3] BTK inhibitors, such as evobrutinib, have entered clinical studies for the treatment of autoimmune diseases.[4]Objectives:Small molecule-induced protein degradation offers a unique approach to target BTK; this approach simultaneously eliminates both BTK kinase activity and BTK-mediated scaffolding interactions in the signalosome. Chimeric Targeting Molecules (CTMs) are small molecules that catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a ubiquitin ligase binding element (“harness”), a linker, and a target binding element (“hook”). NX-5948 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. We examined the activity of NX-5948 in a collagen-induced arthritis model as part of an assessment of its potential as a drug candidate for autoimmune disease.Methods:Cellular degradation of BTK, Aiolos and Ikaros as well as induction of CD69 and CD86 was determined using flow cytometry. Degradation of BTK in CD-1 mice or cynomolgus monkey was determined using flow cytometry analysis. In a collagen-induced arthritis (CIA) model, mice were vaccinated with type II collagen and treated before the onset of symptoms. Serum cytokine and anti-type II collagen antibody levels were determined using Luminex and ELISA, respectively.Results:In human PBMCs, NX-5948 degrades BTK at sub-nanomolar concentrations and inhibits BCR signaling as measured by CD69 and CD86 induction in anti-IgM-stimulated B cells with similar potency. Oral administration of NX-5948 in mice leads to BTK degradation to &lt;10% of baseline levels in circulating and splenic B cells. NX-5948 also promotes potent BTK degradation in cynomolgus monkeys, and it can suppress BTK levels to &lt;10% of baseline levels after a single oral dose as low as 10 mg/kg.Unlike IMiD drugs such as lenalidomide, the CRBN harness of NX-5948 was designed to avoid the degradation of known CRBN neo-substrates Aiolos (IKZF3) and Ikaros (IKZF1). In primary human T cells, NX-5948 induces minimal degradation of Aiolos and Ikaros and does not promote IL-2 secretion suggesting that NX-5948 does not convey IMiD activity associated with agents such as lenalidomide.We examined the activity of NX-5948 in a mouse CIA model compared to that of the BTK inhibitor ibrutinib or dexamethasone as a positive control. In mice treated with NX-5948, symptoms of arthritis were resolved, and a significant reduction in arthritis clinical score was observed. Treatment with NX-5948 resulted in a reduction in anti-type II collagen titer and serum levels of the pro-inflammatory cytokine IL-6. Treatment with NX-5948 yielded superior anti-inflammatory activity relative to ibrutinib and similar activity to dexamethasone. Treatment with NX-5948 was well-tolerated and, unlike dexamethasone, did not promote body weight loss.Conclusion:Degradation of BTK by NX-5948 shows robust activity in a CIA model compared to existing agents tested as controls. These findings provide support for further investigation of NX-5948 in additional models of autoimmune disease to inform plans for clinical development.

Surface Proteomics Reveals CD72 as a Target for In Vitro-Evolved Nanobody-Based CAR-T Cells in KMT2A/MLL1-Rearranged B-ALL.

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861.

Proteolysis Targeting Chimeras for BTK Efficiently Inhibit B-Cell Receptor Signaling and Can Overcome Ibrutinib Resistance in CLL Cells.

Proteolysis targeting chimeras (PROTACs) are small molecules that form ternary complexes between their target and E3 ligase, resulting in ubiquitination and proteasomal degradation of the target protein. Using our own designed Bruton’s tyrosine kinase (BTK) PROTAC compounds, we show herein efficient BTK degradation in chronic lymphocytic leukemia (CLL) cells. The reversible non-covalent compound (NC-1) was the most potent and therefore we focused on this PROTAC to investigate its subsequent effects on the BCR pathway. NC-1 decreased baseline BTK phosphorylation as well as activation of BTK and other signaling molecules downstream of the BCR pathway, following IgM engagement. These effects were also obtained in samples from CLL patients with clinical resistance to ibrutinib and mutations at C481. NC-1 treatment further decreased baseline CD69 surface levels, completely abrogated its upregulation following IgM activation, decreased CLL cells migration toward SDF-1 and overcame stromal anti-apoptotic protection. In conclusion, our results indicate that targeting BTK using the PROTAC strategy could be a potential novel therapeutic approach for CLL.