Targeting Venetoclax-Resistant CLL By Bcl-XL Degradation

Abstract Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in adults and is associated with resistance to apoptosis. Clinical responses are dramatically improved with the use of targeted therapies such as venetoclax, a selective inhibitor of the anti-apoptotic protein Bcl-2, compared to chemoimmunotherapy. However, some patients can ultimately develop resistant CLL. Bcl-xL overexpression has been implicated in multiple mechanisms of venetoclax resistance, representing a switch that confers cell survival despite Bcl-2 inhibition. Mechanisms of venetoclax resistance include mutated Bcl-2 (ex. G101V), switches in survival dependence to alternative BCL2 family members, and microenvironmental interactions (Reviewed in Bose et al., 2017). Moreover, disease progression on venetoclax is accelerated in CLL patients with aberrant p53 , and our laboratory identified Bcl-xL as one of the main contributors to the oncogenic abilities of mutant p53.Thus, Bcl-xL is a promising target for re-sensitizing CLL to apoptosis, but since it is crucial for platelet viability, direct targeting has not been clinically successful. New Bcl-xL PROTAC degraders provide an exciting opportunity to target Bcl-xL in venetoclax-resistant CLL. Due to differences in the expression of VHL E3 ligase in cancer cells versus platelets, these degraders have shown potency and selectivity to target this protein for degradation in multiple Bcl-xL-dependent malignancies (He, Y. et al., 2020). Our results show that exposing primary CLL samples (n=4) from treatment-naïve patients to the Bcl-xL degrader DT2216 is associated with apoptosis at concentrations known to be non-toxic to platelets (EC50 = 162 nM at 18 hr treatment). Furthermore, the dual Bcl-xL/Bcl-2 degrader PZ18753B was synthesized with enhanced potency against Bcl-xL and Bcl-2 co-dependent cancers. Our results confirm enhanced potency in primary cells from treatment naïve patients with CLL (EC50 = 7.602 nM at 18 hr treatment), compared to DT2216. Also, the degraders exhibit a similar kinetic profile at 10-fold concentrations (n=5, t1/2 = 2.3 hr for 1 uM DT2216, and t1/2 = 3.1 hr for 100 nM PZ18753B). For these results, apoptosis via the mitochondrial pathway was studied by flow cytometry, assessing extracellular exposure of phosphatidyl serine and mitochondrial depolarization by Annexin V and TMRM staining, respectively. Consistently, as confirmed by Western Blot, PZ18753B is more potent at degrading Bcl-xL than DT2216, achieving 50% degradation in RS4;11 leukemic cells at 46 hr treatment at a concentration of 0.86 nM, compared to 7.83 nM for DT2216. Comparing dose-response curves showing apoptosis by AnnexinV/TMRM staining and protein degradation by Western Blot, we confirmed that Bcl-xL degradation precedes cell death. Given that Bcl-xL overexpression confers a survival advantage to CLL cells, we hypothesize that venetoclax-resistant CLL cells can be targeted with the new Bcl-xL degraders. We studied the potency of the degraders against RS4;11 leukemic cells which express wildtype (WT) Bcl-2, or also overexpress G101V mutant Bcl-2. This is one of the mutations associated with venetoclax resistance and, combining data from various cohorts, it was identified in 23% venetoclax-resistant patients (Blombery et al., 2019 and 2020; Tausch et al., 2019; Weiss et al., 2019). PZ18753B induced apoptosis of RS4;11 Bcl-2 WT cells within 46 hrs (EC50 = 11.1 nM) as well as in RS4;11 cells expressing Bcl-2 G101V mutation (EC50 = 109.1 nM). PZ18753B degraded both Bcl-xL and Bcl-2 in RS4;11 WT cells effectively. In RS4;11 Bcl-2 G101V cells, it did not affect Bcl-2 but retained potent Bcl-xL degradation (86% vs 75% at 1 uM in WT vs mutant Bcl-2 cell lines, respectively). Interestingly, DT2216 did show ability to degrade G101V mutant Bcl-2 at the highest concentrations tested (Ex. 36% and 33%, respectively, at 1 uM). We also demonstrated that CLL cells from chemo-naïve patients depend primarily on Bcl-2 for survival, as measured by their responses to BH3 mimetic peptides in a BH3 profiling assay. However, venetoclax-resistant CLL cells undergo a shift in dependence to alternative Bcl-2 family proteins, such as Bcl-xL and Mcl-1, as a mechanism for resistance to apoptosis. Thus, resistant CLL represents an excellent setting in which to continue testing the efficacy of these potent Bcl-xL degraders, to overcome resistance to Bcl-2 inhibitors. Disclosures Zheng: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhou: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhang: Dialectic Therapeutics: Other: Co-founder and stakeholder. Co-inventor of Bcl-xL PROTACs disclosed in this study.. Wierda: Oncternal Therapeutics, Inc.: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Xencor: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; GSK/Novartis: Research Funding; Janssen: Research Funding; Loxo Oncology, Inc.: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; Karyopharm: Research Funding; Miragen: Research Funding; Cyclacel: Research Funding; AstraZeneca: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Jain: Incyte: Research Funding; Servier: Honoraria, Research Funding; Precision Biosciences: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Beigene: Honoraria; Cellectis: Honoraria, Research Funding; Pfizer: Research Funding; Fate Therapeutics: Research Funding; Aprea Therapeutics: Research Funding; Genentech: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria, Research Funding; TG Therapeutics: Honoraria; Janssen: Honoraria; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Woyach: AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; Gilead Sciences Inc: Other: Data & Safety; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy.

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Phosphorylation of Receptor Tyrosine Kinase ROR1 At Tyrosine 641, 646 and Serine 652 residues Might Be of Importance for the Survival of CLL Leukemic Cells

Blood ◽

10.1182/blood.v120.21.3885.3885 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3885-3885

Author(s):

Mohammad Hojjat-Farsangi ◽

Amir Hossein Daneshmanesh ◽

Martin Norin ◽

Åsa Sandin ◽

Abdul Salam Khan ◽

...

Keyword(s):

Amino Acid ◽

Tyrosine Kinase ◽

Western Blot ◽

Tyrosine Kinases ◽

Conflicts Of Interest ◽

Annexin V ◽

Kinase Domain ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Tyrosine Kinase Domain

Abstract Abstract 3885 Background: Receptor tyrosine kinases (RTK) play crucial roles for different normal cellular processes as cell proliferation/differentiation, apoptosis and survival, as well as for the malignant phenotype of many types of cancer. ROR1, as a member of twenty RTKs families, has important functions during normal embryogenesis. ROR1 has shown to be overexpressed in chronic lymphocytic leukemia (CLL), mantle cell lymphoma and other hematological malignancies, as well as in solid tumors. ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. In this study, we investigated the effects of an anti-ROR1 mAb against the extracellular CRD domain for specific dephosphorylation at the tyrosine kinase domain of ROR1 in CLL cells. The CRD domain contains a frizzle receptor, which is considered to be the ligand-binding region for e.g. Wnt5a. Wnt5a has been suggested to stimulate growth of CLL cells. Aims: To investigate phosphorylation of tyrosine and serine residues, within the tyrosine kinase domain of ROR1, of importance for survival of CLL cells. Methods: Bioinformatic analysis of the ROR1 structure revealed that three amino acid residues in the tyrosine kinase domain might be critically phosphorylated. Based on this prediction, a 19 amino acid long peptide, phosphorylated at two tyrosine (tyrosine 641, 646) and one serine (serine 652) residues was designed and used for immunization of rabbits. An anti-phospho-ROR1 (pROR1) polyclonal antibody (pAb) with high titers of anti-pROR1 pAb was purified, using phospho-peptide affinity chromatography. The specificity of anti-pROR1 pAb was determined by ELISA, immunoprecipitation (IP) and western blot experiments. An anti-ROR1 mAb (IgG) (CRD 1D8 clone) was used to analyse the effects on ROR1 phosphorylation in CLL cells at tyrosine 641, 646 and serine 652 residues preceding apoptosis. ROR1 phosphorylation was investigated by western blot and IP of ROR1 probed with anti-pROR1 pAb, from untreated and CLL cells treated with the anti-CRD 1D8 mAb. Quantitative intracellular staining of ROR1 by flowcytometry in time kinetics experiment after treatment with anti-CRD 1D8 mAb was also used to check phosphorylation of ROR1. Annexin V/PI staining (flowcytometry), MTT assay, PARP and caspase 8 cleavage as well as MCL-1 protein (western blot) were used for detection of apoptosis. To investigate phosphorylation and localization of 64–130 kDa ROR1 isoforms in various compartments of CLL cells, lysates were prepared from the nucleus and cytoplasmic proteins of CLL cells. Results: Two tyrosine (641, 646) residues and one serine (652) residue of the tyrosine kinase domain were phosphorylated in CLL cells. As previously described (Mellstedt et al, Abstract No: 1771, 53th ASH annals meeting, 2011), the 64, 105 and 130 kDa ROR1 isoforms were shown to be constitutively phosphorylated at tyrosine and serine residues in CLL leukemic cells. Treatment of CLL cells with an anti-ROR1 mAb against the CRD domain induced rapid dephosphorylation of ROR1 at tyrosine 641, 646 and serine 652 residues within 20 min and gradually increased up to 4 hours. The phosphorylated 64 kDa ROR1 isoform was localized to the nucleus of CLL cells and probably represents an intracellular part of ROR1, while the ROR1 130 kDa isoform was presented both in cytoplasm and nucleus of CLL cells. Conclusion: Our data show that the ROR1 molecule is phosphorylated at tyrosine 641, 646 and serine 652 residues. The presence of 64 and 130 kDa ROR1 isoforms in the nucleus of CLL cells may suggest a role of these isoforms as transcription factors. Collectively, the data might suggest that phosphorylated ROR1 may be an important protein for the growth of CLL cells as well as an interesting structure to target in a therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

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Cytosolic Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Blood ◽

10.1182/blood.v106.11.2108.2108 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2108-2108

Author(s):

Eva Gine ◽

Marta Crespo ◽

Ana Muntanola ◽

Emili Montserrat ◽

Francesc Bosch

Keyword(s):

Functional Status ◽

Annexin V ◽

Histone H1 ◽

Mitochondrial Pathway ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Dependent Manner ◽

Immunofluorescence Analysis ◽

Cytochrome C Release ◽

Nuclear Histone

Abstract Recently, it has been shown that nuclear histone H1.2 is released into cytoplasm when apoptosis is induced by DNA double-strand breaks (DSB’s), this process being dependent on p53 functional status. In addition, cytosolic histone H1.2 induces cytochrome C release in a Bak-dependent manner. Thus, cytosolic histone H1.2 release represents a new mechanism that links DSB’s with activation of the apoptotic mitochondrial pathway. Against this background, we analyzed the release of histone H1.2 in the cytosol of purified CLL cells during apoptosis induced by fludarabine (F), mitoxantrone (M), etoposide, or ionizing radiation. In addition, the presence of histone H1 was correlated with p53 functional status. Cell viability and analysis of apoptosis were investigated by annexin V/PI staining and FACscan analysis. The presence of histone H1 and H1.2 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 (Upstate) and anti-histone H1.2 (Abcam) antibodies. Histone H1 traffic was also evaluated by using immunofluorescence analysis in CLL cells suspensions. FISH analysis was used to select samples with (n=3) or without (n=6) p53 deletion, and activation of p53 after treatment was assessed by Western Blot. In cases without p53 deletion, increased apoptosis was observed under all stimuli, the FM combination being the most effective. In such cases, histone H1.2 release was apparent 6 hours after the onset of irradiation or pharmacologic treatments, progressively increasing up to 24 hours. In contrast, cases with p53 deletion displayed a low cytotoxic effect upon different treatments. Interestingly, no p53 activation or histone H1.2 release into cytosol was observed. These results were also confirmed by immunofluorescence analysis, in which histone H1.2 was only visible in the cytosol of non-deleted p53 cases. These results demonstrate that, upon drug or irradiation exposure nuclear histone H1.2 is released into the cytoplasm of CLL cells in a p53-dependent manner. This suggests that, in CLL, histone H1.2 traffic contributes to the apoptosis induced by DSB’s and to drug resistance in cases with p53 deletion.

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Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Blood ◽

10.1182/blood.v110.11.1141.1141 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1141-1141

Author(s):

Eva Gine ◽

Marta Crespo ◽

Ana Muntañola ◽

Eva Calpe ◽

Maria Joao Gomes ◽

...

Keyword(s):

Annexin V ◽

Genetic Alterations ◽

Mitochondrial Pathway ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Dependent Manner ◽

Cytochrome C Release ◽

Dna Damaging Agents ◽

Genetic Abnormalities ◽

P53 Status

Abstract Cytosolic release of histone H1.2 has been described as a new apoptogenic mechanism induced by DNA damage that results in cytochrome C release and activation of the apoptotic mitochondrial pathway. Primary tumoral CLL cells from 25 patients were investigated for histone H1.2 cytosolic release after treatment with genotoxic (fludarabine, mitoxantrone, etoposide, or X-ray radiation) and non-genotoxic (dexamethasone) agents. Cases were analyzed for the presence of poor-risk genetic alterations, particularly deletions at 17p13 and 11q22. Histone H1.2 release was correlated with the presence of genetic abnormalities and with the best clinical response obtained with standard treatments. FISH analysis, cell viability measured by annexin V binding, Western Blot studies and inmunofluorescence techniques with confocal spectral microscopy were also employed. DNA-damaging agents induced H1.2 release in a p53-dependent manner, which was confirmed by the lack of H1.2 release in p53-deleted cases. Non DNA-damaging agents induced release of H1.2 in both p53-deleted and non-deleted CLL cases. Moreover, nuclear H1.2 release was observed after genotoxic and non-genotoxic treatment independently of ATM function. From the clinical standpoint, the lack of histone H1.2 release correlated with resistance to genotoxic treatment. In CLL cells, histone H1.2 traffic was dependent on the p53-status after genotoxic treatment, but was also inducible after treatments acting independently of p53. In contrast, histone H1.2 release seemed not to be dependent on ATM function. Nuclear histone H1.2 release appears to be an important element in apoptosis induction in CLL, particularly in cases with abnormal p53 function resistant to conventional treatment.

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IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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Fcgammariib Expression As a Prognostic Marker in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.1727.1727 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1727-1727

Author(s):

Rosa Bosch ◽

Gerardo Ferrer ◽

Eva Puy Vicente ◽

Alba Mora ◽

Rajendra N. Damle ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Clinical Outcome ◽

B Cell ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Disease Stage ◽

Advanced Stage ◽

Fcγ Receptor ◽

Expression Levels

Abstract BACKGROUND The Fcγ receptor IIb (FcγRIIb) is an inhibitory Fcγ receptor that suppresses B-cell activation when coligated with B-cell antigen receptor (BCR). Previous studies from our group indicate that the ability of the FcγRIIb to inhibit BCR signaling after coligation is attenuated in Chronic Lymphocytic Leukemia (CLL). Furthermore, in contrast to what has been described in normal murine B-cells, stimulation of the FcγRIIb alone induces proliferation of CLL cells. However, the correlation between FcγRIIb expression, immunophenotypic characteristics, and clinical variables in patients with CLL has not been studied. AIM The aim of this study was to correlate the expression of FcγRIIb on leukemic cells from previously untreated CLL patients and its immunophenotypic features and clinical parameters. METHODS The study population included 112 patients with untreated CLL for whom cryopreserved peripheral blood samples were available before treatment. The diagnosis was based on IWCLL 2008 criteria. The median patients' follow up was 57.92 months (range: 2.23-439.78 months). FcγRIIb expression levels were determined by flow cytometry on CD5+/CD19+ CLL cells using a specific Alexa488-conjugated murine mAb specific for human FcγRIIb. The following combinations were assessed: FcγRIIb/CD38/CD19/CD5, FcγRIIb/CD49d/CD19/CD5, and FcγRIIb/CD69/CD19/CD5. Results were expressed as the ratio between the MFI for FcγRIIb and the MFI for the corresponding isotype (MFIR). FcγRIIb expression levels were correlated with: i) expression of CD49d, CD38 and CD69, ii) clinico-biological characteristics, and iii) clinical outcome. Differences of FcγRIIb expression on dichotomized clinicopathological variables were assessed with Mann Whitney test. Kaplan-Meier survival and Cox regression analysis were performed to evaluate the correlation of FcγRIIb expression with clinical outcome. Best cut-offs for overall survival (OS) and treatment-free survival (TFS) were determined by ROC curves. RESULTS All CD5+CD19+ leukemic cells samples expressed FcγRIIb. However, FcγRIIb expression levels markedly varied between patients (median MFIR: 45.8; interquartile range: 14.9-76.6; 5th -95th percentile: 17.15-111.4). FcγRIIb expression was significantly higher in patients who had high (≥30%) CD49d expression than in those with low (<30%) CD49d expression (p =0.009). No correlation was observed between FcγRIIb expression and age, disease stage, IGHV mutational status or chromosomal abnormalities analyzed by fluorescence in situ hybridization, ZAP70 and CD38. Furthermore, within individual clones, FcγRIIb expression levels were higher on CD38+ or CD49d+ cells than on CD38- or CD49d- cells, respectively (median MFIR: 49.05 vs. 36.72, p =0.001 for CD38+ vs. CD38- cells; and 58.87 vs. 35.55, p <0.001 for CD49d+ vs. CD49d- cells). In univariate analysis, low FcγRIIb expression levels (MFIR< 26.67) were associated with shorter OS (HR 4.01, 95%CI 1.15-13.90, p=0.029), together with older age, advanced stage, and expression of CD38 and CD49d. Advanced stage, unmutated IGHV, and CD38, CD49d and ZAP-70 expression were also associated significantly with shorter TFS. Thus, patients with higher levels of FcγRIIb had better survival than those with lower levels (Log rank test, p = 0.018). A multivariate analysis adjusted for FcγRIIb expression, age, disease stage, CD38, and CD49d identified older age (≥65 yrs) (HR 150.76, 95%CI 5.39-4212.42, p =0.003), low FcγRIIb expression (HR 111.91, 95%CI 6.71-1866.97, p =0.001), advanced stage (B/C) (HR 17.44, 95%CI 1.45-210.24, p =0.024) and CD38 expression (HR 5.02, 95%CI 1.01-25.18, p =0.050) as independent predictors for shorter OS. CONCLUSIONS In this study, FcγRIIb expression on leukemic cells from untreated patients with CLL was found to be an independent prognostic marker for OS, overcoming the prognostic value of CD49d, which is consistent with the key role of the FcγRIIb in the pathogenesis of CLL. Further analysis aimed at validating this observation and to better understand the functional cooperation of FcγRIIb with other molecules, particularly CD49d, are warranted. These studies could open a new venue in CLL treatment. Disclosures Gorlatov: MacroGenics: Employment. Sierra:Novartis: Research Funding; Celgene: Research Funding; Amgen: Research Funding.

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Effect of ROR1-targeting small molecules on chronic lymphocytic leukemia (CLL) cells.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.6557 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 6557-6557 ◽

Cited By ~ 1

Author(s):

Hakan Mellstedt ◽

Salam Khan ◽

Jan Vågberg ◽

Styrbjörn Byström ◽

Johan Schultz ◽

...

Keyword(s):

Tyrosine Kinase ◽

Western Blot ◽

Small Molecules ◽

High Throughput Screening ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Parp Cleavage ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Substrate Peptide

6557 Background: There is a great interest to develop targeting drugs for CLL, both MAbs and small molecules, to improve the outcome for the disease. ROR1, a receptor tyrosine kinase, is overexpressed on CLL cells but not on normal cells. ROR1 is constitutively phosphorylated and siRNA transfection induces leukemic cell death. The aim of the study is to produce small molecules inhibiting the cytoplasmic tyrosine kinase activity of ROR1, which could induce specific killing of leukemic cells. Methods: A high-throughput screening assay in 384-format has been developed measuring phosphorylation of a substrate peptide by human recombinant intracellular ROR-1 kinase domain. A collection of 80.000 small molecules has been screened generating two chemical series. Novel leads have been synthesized and structure-activity-relationship has been developed using the assay. Results: Three compounds were selected (KAN0173631, KAN0438063, KAN0438175T). Freshly isolated leukemic CLL cells were used as targets in addition to PBMC from healthy donors to test cytotoxicity. The three compounds induced specific apoptosis of CLL cells (Annexin V/PI and MTT) both at 24 and 48h. Efficacy index (EI), i.e. killing of leukemic cells in relation to normal PBMC was favourable. The most promising drug KAN0438063 had an EI of 40 i.e. killed 40 times more CLL cells than PBMC at an IC50 of 10µM. The compounds induced PARP cleavage and cleavage of caspases 8 and 9 as well as down regulation of Mcl-1 and Bcl-xl (Western Blot). ROR1 was dephosphorylated (Western Blot). The selective apoptotic effect was compared to other small molecules targeting non-ROR1 structures in CLL (PCI-32765, CAL-101, R406, R788, STK-156485, STK-156133) and our compounds were significantly more effective (EI) (p<0.001). Conclusions: We have developed effective and selective series of compounds targeting ROR1 with promising ADMET properties. ROR1 targeting small molecules might also be effective for other tumor cells expressing ROR1 as has been noted for e.g. pancreatic carcinoma cells. Our model molecules will be further optimized and tested in animal tumor models. These molecules represent the first small molecules targeting ROR1 – a “survival factor” in CLL.

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Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v116.21.778.778 ◽

2010 ◽

Vol 116 (21) ◽

pp. 778-778

Author(s):

Zhihong Zeng ◽

Yuexi Shi ◽

Twee Tsao ◽

Yihua Qiu ◽

Steven M. Kornblau ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Signaling Pathways ◽

Research Funding ◽

Kinase Inhibitor ◽

Annexin V ◽

Mtor Signaling ◽

Leukemic Cells ◽

Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

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Indoleamine 2,3-Dioxygenase Mediates Survival of Chronic Lymphocytic Leukemia B Cells through Aryl Hydrocarbon Receptor By Inducing Mcl1

Blood ◽

10.1182/blood-2020-142345 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 19-19

Author(s):

Claudio Giacinto Atene ◽

Rossana Maffei ◽

Stefania Fiorcari ◽

Silvia Martinelli ◽

Patrizia Zucchini ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Aryl Hydrocarbon Receptor ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Strong Increase ◽

Indoleamine 2,3 Dioxygenase ◽

Aryl Hydrocarbon

Introduction: Chronic lymphocytic leukemia (CLL) is a dynamic disease in which monoclonal B cells proliferate within the pseudo-follicular centers in lymphoid organs and then they accumulate due to an intrinsic defect of apoptosis. Leukemic cells are considered as "addicted to the host" since extrinsic signals from the microenvironment strongly influence the establishment of a progressive immunosuppression for malignant cell growth and survival. The cytoplasmic enzyme indoleamine 2,3-dioxygenase (IDO) mediates the conversion of the essential amino acid tryptophan (Trp) into metabolic byproducts such as kynurenine (Kyn). Kyn and other secondary metabolites are endogenous activators of the aryl hydrocarbon receptor (AHR), a ligand-controlled transcription factor that mediates cellular responses to toxins or endogenous ligands. The IDO-Kyn-AHR axis plays important roles in carcinogenesis and cancer progression. The mechanisms that promote inflammation around tumor tissues and determine immune tolerance consist in Trp depletion, which induces T cell apoptosis, and in Kyn-mediated AHR activation that inhibits effector T cells and promotes regulatory T cells differentiation. IDO protein is expressed in human hematologic malignancies and its level is correlated with a poor prognosis and chemoresistance. The IDO activity, measured as the Kyn/Trp ratio, was reported to be increased in CLL cases comparing to normal controls. Aim: We wondered to characterize the expression of IDO and AHR in CLL patients and to dissect the biological function of the IDO-Kyn-AHR axis. Methods: Gene transcription and protein expression were evaluated by real time PCR and western blot. Enzymatic activity was assessed through ELISA. Survival was measured with PI/annexin V assay. Overexpression and silencing of target genes was obtained by nucleofection. Results: Firstly, we observed that CLL cells expressed both IDO and AHR at variable levels. Moreover, we found that several microenvironmental signals such as IFNγ, LPS, anti-IgM, CpG oligo DNA, CD40L and TNFα were able to up-regulate IDO and AHR mRNA and protein. To characterize the pathways able to mediate IDO expression, we stimulated CLL cells with IFNγ and CD40L. Using ruxolitinib, an inhibitor of JAK-STAT pathway, we found that IFNγ induced IDO through STAT1 signaling. Again, CD40L stimulation determined IDO overexpression through the non-canonical NF-kB pathway, as assessed by treating cells with NF-κB inducing kinase inhibitor, NIK SMI1. We also confirmed that IFNγ-treated CLL cells were able to produce a functional IDO enzyme by measuring Kyn production and Trp consumption by ELISA. The strong increase in the Kyn/Trp ratio induced by IFNγ was significantly reduced by ruxolitinib treatment. To verify if Kyn produced by CLL cells could act through an autocrine loop on AHR, leukemic cells were treated with Kyn. We observed that Kyn mediated AHR translocation from the cytoplasm to the nuclei, inducing its activation as assessed by up-regulation of CYP1A1, a known AHR target gene. Of interest, we found that Kyn treatment improved CLL cells survival. Analyzing the anti-apoptotic proteins of the Bcl2 family after Kyn treatment, we found the induction of Mcl1, that was affected by adding CH-223191, an antagonist of AHR. Moreover, we transfected CLL cells with an IDO vector. The up-regulation of IDO increased CLL cells survival through the induction of Mcl1. Accordingly, when CLL cells were silenced for AHR, we observed a reduction of their survival. Conclusion: Our data demonstrate the constitutive expression of IDO and AHR in CLL cells. Furthermore, the tumor microenvironment promotes the induction of IDO and AHR through a complex signaling crosstalk with leukemic cells. Our findings underline that IDO-Kyn-AHR axis is active in CLL cells and promotes Mcl1 expression, sustaining the survival of CLL cells. Disclosures Luppi: Gilead Sci: Consultancy, Speakers Bureau; MSD: Consultancy; Sanofi: Consultancy; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy; Novartis: Consultancy, Speakers Bureau. Marasca:Gilead Sci: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria.

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Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Blood ◽

10.1182/blood-2021-146539 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1318-1318

Author(s):

Dipabarna Bhattacharya ◽

Jani Huuhtanen ◽

Matti Kankainen ◽

Tapio Lönnberg ◽

Cassandra M Kerr ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Healthy Controls ◽

Advisory Committees ◽

Cell Clones ◽

Tcr Repertoire

Abstract Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL. Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure) Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively. Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data. To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens. To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response. Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes. Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. Figure 1 Figure 1. Disclosures Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

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Human Macrophages Phagocytose Rituximab Opsonised Leukemic Cells Via CD16, CD32 and CD64 but Do Not Mediate ADCC.

Blood ◽

10.1182/blood.v108.11.2507.2507 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2507-2507

Author(s):

Josee Golay ◽

Marzia Leidi ◽

Giuseppe A. Palumbo ◽

Martino Introna

Keyword(s):

Complement Activation ◽

Xenograft Model ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Therapeutic Activity ◽

Blood Monocytes ◽

Response Curves ◽

Human Macrophages

Abstract Rituximab (Mabthera®) is a chimeric monoclonal IgG1 antibody with therapeutic activity in non-Hodgkin B lymphomas (B-NHL) and B-Chronic Lymphocytic Leukemia (B-CLL). We have recently obtained evidence, using a bulky lymphoma xenograft model in nude mice, that both complement and macrophages are required for the therapeutic activity of rituximab. In order to further investigate the tumor cell killing potential of macrophages and its modulation by different factors, including complement, we have set up in vitro experiments with purified macrophage populations. Human macrophages were obtained from purified peripheral blood monocytes cultured for 4 days in presence of 20% FCS and 20 ng/ml M-CSF. FACS analysis confirmed the phenotype of these cells including CD11b and FcγRs expression (CD16, CD32, CD64). Phagocytosis assays were then carried out with CLL cell as targets in presence or absence of increasing concentrations of rituximab. Phagocytosis was evaluated by counting under an inverted microscope the stained cytospin preparations. From 9.8% to 60.8% of macrophages engulfed at least one tumor target cell in a series of 24 experiments (mean 29.7%± 18.3%). Control irrelevant IgG1k monoclonal antibodies (anti-erbB2 trastuzumab and anti-EGFR cetuximab) did not mediate phagocytosis, and rituximab did not lead to ingestion of CD20 negative cells, demonstrating the specificity of the assay. Phagocytosis was already maximal at around 0.1 μg/ml rituximab concentration. In contrast complement activation required Mab concentration of at least 1 μg/ml. Thus phagocytosis, like ADCC, is active at about 10 fold lower MAb concentrations than complement triggering. Levels of CD20 expression on targets did not significantly affect phagocytosis. The role of different FcγRs was also investigated by addition 5 μg/ml blocking antibodies to CD16, CD32 and CD64. All 3 blocking Mabs reduced significantly phagocytosis (by 45%, 42% and 40% respectively with respect to control). Inhibition increased to 64% in presence of all 3 antibodies. Since previous data had suggested a role of the Val/Phe polymorphism at position 158 of CD16A in the clinical response of lymphoma patients to rituximab as well as in NK-mediated ADCC, we investigated whether this polymorphism also affected phagocytosis. No significant differences in dose response curves were observed using macrophages from either Val-Val or Phe-Phe homozygotes. Perhaps surprisingly, concomitant complement activation induced by addition of human serum did not increase phagocytosis. Whether human macrophages can also mediate antibody dependent cellular cytotoxicity (ADCC) was also studied. CLL or BJAB cells were labeled with Calcein-AM and ADCC measured as released fluorescence after 4 hours at 37°C. Macrophages were unable to mediate ADCC in presence of rituximab even following treatment with IFNγ (100 U/ml) for 48 hours. We conclude that macrophages efficiently mediate phagocytosis but not ADCC in presence of low concentrations of rituximab.

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