Chronic Lymphocytic Leukemia Cell Adhesion Research Articles

The Role of the Microenvironment and Cell Adhesion Molecules in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy whose progression largely depends on the lymph node and bone marrow microenvironment. Indeed, CLL cells actively proliferate in specific regions of these anatomical compartments, known as proliferation centers, while being quiescent in the blood stream. Hence, CLL cell adhesion and migration into these protective niches are critical for CLL pathophysiology. CLL cells are lodged in their microenvironment through a series of molecular interactions that are mediated by cellular adhesion molecules and their counter receptors. The importance of these adhesion molecules in the clinic is demonstrated by the correlation between the expression levels of some of them, in particular CD49d, and the prognostic likelihood. Furthermore, novel therapeutic agents, such as ibrutinib, impair the functions of these adhesion molecules, leading to an egress of CLL cells from the lymph nodes and bone marrow into the circulation together with an inhibition of homing into these survival niches, thereby preventing disease progression. Several adhesion molecules have been shown to participate in CLL adhesion and migration. Their importance also stems from the observation that they are involved in promoting, directly or indirectly, survival signals that sustain CLL proliferation and limit the efficacy of standard and novel chemotherapeutic drugs, a process known as cell adhesion-mediated drug resistance. In this respect, many studies have elucidated the molecular mechanisms underlying cell adhesion-mediated drug resistance, which have highlighted different signaling pathways that may represent potential therapeutic targets. Here, we review the role of the microenvironment and the adhesion molecules that have been shown to be important in CLL and their impact on transendothelial migration and cell-mediated drug resistance. We also discuss how novel therapeutic compounds modulate the function of this important class of molecules.

Abstract 2966: Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells

Abstract Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2966.

Integrin Signaling Pathway Is Crucial for the Recently Divided Fraction of CLL Cells and Facilitates Cell Migration in a Disease Progressive Manner

Chronic lymphocytic leukemia (CLL) cells can be subdivided into fractions based on the time since cell birth/division. The fraction of CLL B cells bearing a CXCR4DimCD5Bright phenotype divided recently (proliferative fraction, PF), while those cells that divided earlier and are quiescent bear a CXCR4BrightCD5Dim phenotype (resting fraction, RF). Prior studies suggest that cells of the PF are recent emigrants of solid tissue, whereas cells of the RF did so earlier and are most adept at homing to lymphoid niches. Here we report gene expression and functional analyses of the PF and RF that address the signaling pathway most relevant for the PF and the relative abilities of the PF and RF to migrate using the pathway. When analyzing genes significantly differentially expressed (DEGs) between PF and RF of 26 CLL patients (13 U-CLL and 13 M-CLL), we found 198 genes upregulated in the PF and 88 in the RF. Using Ingenuity Pathway Analysis (IPA), the DEGs of the PF overlapped best with the integrin signaling pathway (Z-score = 2.887). Moreover, of the other identified pathways with Z scores > 2.0, each impacted integrin expression and function (upregulated: Actin Nucleating by ARP-WASP Complex and Regulation of Actin-based Motility by Rho; downregulated: RhoGDI signaling). To validate the transcriptomic data, mRNA and protein levels of various upregulated integrin subunits were confirmed by Q-PCR and cell surface staining by flow cytometry. When comparing the DEGs between the PF and the RF in U-CLL vs M-CLL patients, a core of 144 genes were shared by the two and 358 were unique to U-CLL and 35 to M-CLL. Notably, IPA indicated that the shared genes overlapped with the integrin signaling pathway and the U-CLL unique genes resembled pathways used by integrins to carry out their functions, such as migration. When the 3 sets of DEGs were subjected to Upstream Regulator analysis using IPA, multiple cytokines were identified as potential regulators of the DEGs shared by U-CLL and M-CLL (Z scores > 2.0) clones, whereas for the DEGs unique to the PF of U-CLL, only IL-4 was identified (Z scores = 2.6). To corroborate the above, we cultured CLL B cells for 3 days with the of putative upstream regulators of the shared and U-CLL unique DEGs. For the former, we found that [1] IFNα upregulated CD11c in all CLL, [2] IL-1 and IL-2 upregulated CD11a in all CLL, and [3] IL-1, IL-2, IFNα and IFNγ upregulated CD29 in M-CLL only. When testing a regulatory role for IL-4 in the U-CLL unique genes, we found that IL-4 upregulated CD11a, CD11c, and CD18 in U-CLL (P < 0.05), but not in M-CLL. Finally, we evaluated several functions mediated by integrin molecules. First, we tested the binding of the PF and RF to fibronectin, an important step in cell migration. This indicated that the PF bound better to fibronectin than RF (P < 0.01), and led to extended survival (P < 0.05). Consistent with this, co-culture with anti-CD49d antibody significantly blocked CLL cell adhesion to fibronectin (P < 0.01). Next, we addressed CLL cell migration. In vitro migration studies indicated that PF cells migrated significantly better than the RF with (P < 0.0001) or without (P < 0.001) CXCL12/SDF-1. In line with this, 24-hours after transferring PF and RF into NSG mice, significantly more PF than RF cells were found in the spleen (P < 0.05). Notably, the ability to more rapidly enter solid tissue associated with the rate of disease progression of individual patients (P < 0.001). Since these were unexpected findings, we questioned if sorting PF and RF altered their ability to migrate in vitro. Based on the analysis of 16 patients (U-CLL = 8, M-CLL = 8), simultaneous preincubation of CLL B cells with mAbs to CD5 plus CXCR4 did not change spontaneous nor CXCL12/SDF-1-mediated migration. Collectively, our findings indicate that integrins and the integrin signaling pathway are key elements in the biology of the recently divided fraction of CLL cells. Moreover, those DEGS that are shared by U-CLL and M-CLL cells and those unique to U-CLL are regulated by distinct upstream influences, with IL-4 standing out as a crucial cytokine for the development of the U-CLL PF. Finally, in vitro and in vivo studies suggest a key role for the integrin pathway in CLL migration, and pinpoint, surprisingly, the PF, not the RF, as the better migrater and document that the rate of migration correlates with disease activity.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

Elastin MIcrofibriL INterfacer1 (EMILIN-1) is an alternative prosurvival VLA-4 ligand in chronic lymphocytic leukemia.

CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL.

Defining the Biochemical Role of Sialic Acid-Binding Immunoglobulin-like Lectin-6 in Adhesion and Migration in Chronic Lymphocytic Leukemia

Introduction: Sialic acid-binding immunoglobulin-like lectins (Siglec) are a group of lectins that regulate innate and adaptive immune function via glycan recognition. We and others have shown overexpression of Siglec-6, a member of Siglec family, on B cells from patients with chronic lymphocytic leukemia (CLL) compared to normal donor derived B cells. While placental expression of Siglec-6 has been shown to regulate invasion of trophoblast cells by binding to glycodelin, the biochemical role of Siglec-6 in CLL patients is not known. We describe here for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in CLL. Biochemical mechanisms of Siglec-6 mediated cell adhesion and migration through DOCK8 dependent activation of Cdc42 associated with actin polymerization in CLL cells are presented. Further, the physiological relevance of Siglec-6/ DOCK8 axis in CLL cell adhesion and migration is validated using primary CLL patient samples, and genetically engineered loss of function Siglec-6 and DOCK8 mutant MEC1 CLL cell line. These studies thus elucidate the biological role of Siglec-6 in malignant CLL B cells and demonstrate therapeutic opportunities targeting Siglec-6 in CLL.Methods: Flow cytometry was used to analyze surface expression of Siglec-6 and sTn in CLL patients and normal donors. Transwell migration assay was used to assess in-vitro migratory role of Siglec-6. Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. Cdc42 activation was evaluated using a commercial kit which uses specialized PAK1-PBD agarose beads to pull down GTP-bound Cdc42. To study the in-vivo migratory role of Siglec-6, MEC1 CLL cell line or primary CLL cells were blocked with an isotype antibody or Siglec-6 targeted antibody and injected into the tail vein of NSG immunocompromised mice. 24 hrs later, mice were euthanized and spleens and BM were processed followed by flow cytometry analysis to determine the number of human CD45+ cells that have migrated.Results: We confirmed Siglec-6 overexpression on B cells from CLL patients when compared to B cells from normal donors. Interestingly, we also found expression of sTn on bone marrow stromal cells (BMSCs) derived from CLL patients but not healthy donors. Compared to Siglec-6 + CLL cells, Siglec-6 - CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) media containing sTn or sTn + CLL-BMSCs in cell adhesion and trans-well migration assays. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6 + MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6 + MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, which are both downstream targets of DOCK8 involved in cell migration. Further, sTn also promoted actin polymerization, an effect that was compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6 + MEC1 cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane.Conclusions: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. Siglec-6 signals via DOCK8 to mediate sTn ligand dependent actin polymerization. We have also shown that sTn promotes Cdc42 activation and WASP protein recruitment which are both essential for actin polymerization. Moreover, all these effects were prevented by CRISPR/Cas9 mediated knock out of Siglec-6 or DOCK8 in MEC1 CLL cell line. Thus, Siglec-6 represents a CLL-specific target that opens up new therapeutic avenues to target only malignant B-CLL cells. Ongoing studies are focused on determining molecular mechanisms of Siglec-6 mediated regulation of actin polymerization and CLL-BMSC interactions.[This work was supported by NIH-R21 Grant and Pelotonia Idea grants. JN is a recipient of Pelotonia Graduate Fellowship] DisclosuresByrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Calcium-RasGRP2-Rap1 signaling mediates CD38-induced migration of chronic lymphocytic leukemia cells

AbstractCD38 is a transmembrane exoenzyme that is associated with poor prognosis in chronic lymphocytic leukemia (CLL). High CD38 levels in CLL cells are linked to increased cell migration, but the molecular basis is unknown. CD38 produces nicotinic acid adenine dinucleotide phosphate and adenosine 5′-diphosphate-ribose, both of which can act to increase intracellular Ca2+ levels. Here we show that CD38 expression increases basal intracellular Ca2+ levels and stimulates CLL cell migration both with and without chemokine stimulation. We find that CD38 acts via intracellular Ca2+ to increase the activity of the Ras family GTPase Rap1, which is in turn regulated by the Ca2+-sensitive Rap1 guanine-nucleotide exchange factor RasGRP2. Both Rap1 and RasGRP2 are required for CLL cell migration, and RasGRP2 is polarized in primary CLL cells with high CD38 levels. These results indicate that CD38 promotes RasGRP2/Rap1-mediated CLL cell adhesion and migration by increasing intracellular Ca2+ levels.

New players in the Bruton’s tyrosine kinase vs. ibrutinib match

The Bruton’s tyrosine kinase, also known as BTK, is a member of the Tec family kinases with a well-characterized role in B-cell receptor (BCR) signaling and B-cell activation. BTK is able to transmit and amplify several signals that are involved in the complex crosstalk between tumor cells and microenvironment. These signals include cytokines and growth factors, chemokine receptors, antigen receptors as BCR and integrins (1). Chronic lymphocytic leukemia (CLL) cells show an increased expression and activation of BTK that with a chain reaction modulates signaling pathways essential for CLL-cells survival, as the protein kinase AKT, extracellular signal-regulated kinase (ERK) and nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) pathways (2). In addition, BTK is involved in chemokine-mediated migration, homing and adhesion of CLL cells (3). Given the known importance of BCR signaling in CLL and the central role of BTK in this pathway, in the recent years a new drug called ibrutinib has been used in clinical practice to induce inhibition of this kinase. Ibrutinib is a relatively selective, irreversible inhibitor of BTK binding covalently the Cys-481 in the ATP binding domain of the kinase. Inhibition of BTK in CLL cells leads to an impressive delocalization of leukemic cells from the protective tissue microenvironment to the periphery interfering with pathogenic mechanism of recirculation and homing. Several studies demonstrate the considerable clinical success of ibrutinib showing a good safety profile determining an improvement of the quality of life of CLL patients (4).

The Human CD38 Monoclonal Antibody Daratumumab Shows Antitumor Activity and Hampers Leukemia-Microenvironment Interactions in Chronic Lymphocytic Leukemia.

Purpose: To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ chronic lymphocytic leukemia (CLL) subtype.Experimental Design: The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP), or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models.Results: Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo whereas exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38+ MEC2 cell line and CLL patient-derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs, and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by downregulation of the matrix metalloproteinase MMP9.Conclusions: These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL. Clin Cancer Res; 23(6); 1493-505. ©2016 AACR.

Highlights of This Issue

Detection of signals of micrometastatic disease in early breast cancer may improve risk stratification, and serum metabolomic profiles of early breast cancer patients have been shown to be predictive of relapse in a single institution series. Taking samples from a multicentre, phase III adjuvant breast cancer trial in premenopausal women, Hart and colleagues used NMR spectra of preoperative serum samples to build a model that identified women at higher risk of relapse, independent of standard risk factors. Metabolomic analysis has the potential to improve disease staging and prognostication and allow better tailoring of adjuvant therapies.MERTK tyrosine kinase is a potential therapeutic target in acute leukemia. DeRyckere and colleagues developed a series of novel, orally bioavailable MERTK-selective tyrosine kinase inhibitors and now report robust activity mediated by UNC2025, a third-generation compound suitable for clinical development in preclinical acute leukemia models. UNC2025 had antileukemia activity in approximately 30% of 260 freshly isolated leukemia patient samples and had therapeutic activity alone and in combination with cytotoxic chemotherapy in cell line and patient-derived xenografts, suggesting that a large fraction of patients with acute leukemia could benefit from treatment with a MERTK-targeted therapy.CD38 expression is associated with poor prognosis in chronic lymphocytic leukemia (CLL). CD38 is an ectoenzyme that cooperates in migration, adhesion, and invasion through its association with CXCR4, MMP9, and CD49d. To explore its potential as a drug target in CLL, Matas-Céspedes and colleagues analyzed the effect of the anti-CD38 monoclonal antibody daratumumab. The authors demonstrate a dual effect of daratumumab by erradicating the tumor cell both in vitro and in vivo, and by hampering CLL cell adhesion, migration, and homing to secondary organs. These results provide scientific rationale for the use of daratumumab in poor prognosis CD38+CLL.The role of neutrophils in the tumor microenvironment remains controversial. To explore their role in gastric cancer development, Li and colleagues analyzed the distribution and clinical relevance of neutrophils in different microanatomical areas and peripheral blood and assessed the regulation and function of neutrophils by both in vitro and in vivo studies. Their results provided direct evidence supporting the pivotal role of IL-17+ neutrophils in promoting angiogenesis during gastric cancer progression and revealed a fine-tuned collaboration between cancer cells and immune cells in distinct tumor milieus, potentially providing future therapeutic avenues against cancer.

Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia

AbstractInhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity however to induce cell death as monotherapy and are unlikely to eradicate the disease. Acquired resistance to therapy in CLL is often caused by mutations in the response network being targeted, both for DNA damage or BCR signaling pathways. Thus, drugs with dual targeting capacity could offer improved therapeutic value. Here, the potency of CC-115, a novel inhibitor of mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK), was evaluated in primary CLL cells in vitro and in CLL patients. Combined TORK and DNA-PK inhibition in vitro resulted in caspase-dependent cell killing irrespective of p53, ATM, NOTCH1, or SF3B1 status. Proliferation induced by CD40+ interleukin-21 stimulation was completely blocked by CC-115, and CD40-mediated resistance to fludarabine and venetoclax could be reverted by CC-115. BCR-mediated signaling was inhibited by CC-115 and also in CLL samples obtained from patients with acquired resistance to idelalisib treatment. Clinical efficacy of CC-115 was demonstrated in 8 patients with relapsed/refractory CLL/small lymphocytic lymphoma harboring ATM deletions/mutations; all but 1 patient had a decrease in lymphadenopathy, resulting in 1 IWCLL partial response (PR) and 3 PRs with lymphocytosis. In conclusion, these preclinical results, along with early promising clinical activity, suggest that CC-115 may be developed further for treatment of CLL. The trial was registered at www.clinicaltrials.gov as #NCT01353625.

Idelalisib for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma.

Idelalisib is a first-in-class selective oral PI3Kδ inhibitor for the treatment of patients with relapsed chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma, a predominantly elderly population with high comorbidity. The drug promotes apoptosis in primary CLL cells ex vivo, independent of common prognostic markers and inhibits CLL cell homing, migration and adhesion to cells in the microenvironment. Idelalisib has shown efficacy with acceptable safety as monotherapy and combination therapy in relapsed/refractory CLL. Idelalisib has clinical activity in patients with CLL with del(17p). The development of other novel B-cell-targeted agents provides the opportunity to evaluate additional idelalisib treatment combinations for their potential to further improve outcomes in CLL/small lymphocytic lymphoma.

Retention of inside-out VLA-4 Integrin Activation upon B-Cell Receptor Triggering in in-Vitro and in-Vivo Ibrutinib Treated Chronic Lymphocytic Leukemia Cells: Clinical Implication

Background. Ibrutinib (IB), an orally administered Bruton tyrosine kinase (BTK) inhibitor antagonizing B-cell receptor (BCR) signaling, has demonstrated high response rates in chronic lymphocytic leukemia (CLL). Transient lymphocytosis is a common, although variable, clinical feature during IB treatment, which has been related to the efflux of CLL cells from lymphoid organs, due to impairment of microenvironmental interactions. CD49d, a strong negative prognosticator in CLL, is the alpha-chain of the integrin heterodimer CD49d/CD29 (VLA-4), a key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by BCR signals, although the role of BCR as VLA-4 activator in CLL is largely unknown.Aim. To understand whether the inhibition of the BCR signaling by IB exposure of CLL cells affects VLA-4 activation.Methods. The study included 28 refractory/relapsed CLL patients (15 CD49d+, 23 IGHV unmutated, 22 del11q/del17p) treated with IB on a compassionate basis (IB-CLL), and 11 additional CD49d+ CLL cases (7 IGHV unmutated, 1 del17p). Levels of the active VLA-4 were assessed by flow cytometry using an LDV-containing small molecule as VLA-4 specific ligand, along with the conformation sensitive anti-CD29 HUTS-21 mAb, and measuring the VLA-4 receptor occupancy (VLA4-RO) in values ranging from 0.0 (no RO) to 1.0 (100% RO) as in Chigaev et al. (J Biol Chem, 284,14337, 2009). According to this measurement higher RO indicates larger fraction of high affinity VLA-4 receptors. BCR engagement was performed using goat F(ab′)2 anti-human IgM. CD49d, CD29, IgM expression, the phosphorylation state of ERK (pERK) and BTK (pBTK), and Ca++ release were cytometrically analysed. Adhesion assays were performed on VCAM-1-coated slides.Results. CD49d+IB-naïve cells from 18 CLL cases, including 7 pre-treatment IB-CLL, were stimulated with anti-IgM. Although with variability, in all cases cells responded to BCR triggering in terms of increased pERK (mean anti-IgM/control MFI ratio: 1.7), pBTK (mean anti-IgM/control MFI ratio: 1.3), and Ca++ release (mean Ca++ release increase: 11.9%). IgM stimulation resulted in an increased VLA-4 RO (mean 0.52, range 0.40-0.73, vs. mean 0.38, range 0.22-0.63; p=0.0002) in CLL, but not in residual T cells (mean 0.34 vs. 0.35). Consistently, VLA-4-dependent CLL cell adhesion to VCAM-1 increased upon IgM stimulation (mean values of adherent cells/control= 4.6 vs 3.7, p=0.008). IB treatment in vitro ( 1µM, 1 hour) overall impaired BCR signaling, as shown by lower increases of pERK ( mean anti-IgM/control MFI ratio: 1.1), pBTK (mean anti-IgM/control MFI ratio: 1.2) and Ca++ release (mean increase: 7.4%). However, VLA-4 RO increased from a mean of 0.37 (range 0.18-0.65) to 0.47 (range 0.15-0.65) after IgM stimulation (p=0.0007), and a parallel increased adhesion was observed (mean values of adherent cells/control= 2.4 vs 3.7). We then moved to cells collected from IB-CLL on t30 of IB treatment (n=7), which showed slightly decreased (79-86%) CD49d, CD29, and IgM MFI levels compared to pre-treatment cells. Despite an overall impaired BCR signaling (mean Ca++ release increase: 12.4% at pre-treatment, 3.6% at t30), IgM stimulation still increased both VLA-4 RO (mean 0.27, range 0.01-0.45 compared to 0.53, range 0.41-0.84, p=0.010) and CLL cell adhesion. Similarly, cells from IB-CLL on day 60-90 of treatment (n=3) kept showing an increased VLA-4 RO upon IgM stimulation (from 0.14, range 0.10-0.17 to 0.34, range 0.20-0.56), although Ca++ release was relevantly decreased (from 14.8% at pre-treatment to 4.3% at t60-90). Finally, the impact of CD49d expression on the IB-induced lymphocyte redistribution in the 28 IB-CLL was evaluated. Absolute lymphocyte count (ALC) at baseline and after 30-120 days of IB therapy were lower in CD49d+ than CD49d- CLL (see Figure), with a lower median percentage ALC rise from baseline (0% vs +132%, p=0.0044). Consistently, at day t30/t60, >50% reduction of lymph nodes/spleen from pre-treatment stage only occurred in CD49d- CLL (8/13 cases), while CD49d+ CLL usually experienced reductions <50% (11/12 cases).Conclusions. BCR triggering in CLL cells activates VLA-4 via an inside-out pathway at least in part independent to IB blockage of BTK. This observation has clinical implication in the kinetics of ALC, shrinkage of CLL masses, and should be considered in the design of IB therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment with Ibrutinib Inhibits BTK- and VLA-4-Dependent Adhesion of Chronic Lymphocytic Leukemia Cells In Vivo.

Ibrutinib leads to a transient lymphocytosis in patients with chronic lymphocytic leukemia (CLL) that develops within hours of starting drug and is due to the efflux of cells from lymphoid tissues into the blood. We therefore sought to investigate the in vivo effect of ibrutinib on migration and adhesion of CLL cells. Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays. Adhesion of CLL cells to fibronectin was rapidly (within hours) and almost completely inhibited (median reduction 98% on day 28, P < 0.001), while the effect on migration to chemokines was more moderate (median reduction 64%, P = 0.008) and less uniform. Although cell surface expression of key adhesion molecules such as CD49d, CD29, and CD44 were modestly reduced, this was only apparent after weeks of treatment. Stimulation of CLL cells from patients on ibrutinib with PMA, which activates PKC independent of BTK, restored the ability of the cells to adhere to fibronectin in a VLA-4-dependent manner. Finally, the addition of ibrutinib to CLL cells adhered to fibronectin in vitro caused the detachment of 17% of the cells, on average; consisten t with in vivo observations of an increasing lymphocytosis within 4 hours of starting ibrutinib. Inhibition of BTK and VLA-4-dependent adhesion of CLL cells to stroma and stromal components provides a mechanistic explanation for the treatment-induced lymphocytosis and may reduce CD49d-dependent prosurvival signals in the tissue microenvironment.

In Vivo Ibrutinib Abrogates BCR-Dependent Adhesion in Tumor Cells of Patients with Chronic Lymphocytic Leukemia

Ibrutinib, now FDA approved for use in both previously treated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), induces objective clinical responses and is well tolerated (Advani, JCO 2013; Byrd, NEJM 2013). As with other BCR-directed kinase inhibitors, (such as fostamatinib and idelalisib) ibrutinib leads to a treatment-induced transient lymphocytosis that resolves over the first 2-3 months on therapy in patients with CLL. The initial increase in absolute lymphocyte count (ALC) has been shown to be due to the release of tumor cells from the tissue compartments; specifically the lymph nodes, into the peripheral blood (Herman, Leukemia 2014). In vitro ibrutinib reduces the ability of CLL cells to migrate towards chemokines and prevents adhesion of BCR stimulated CLL cells (Ponader, Blood 2012; de Rooij, Blood 2012). We hypothesized that the initial rise in ALC is due to inhibition of CLL cell adhesion. We tested this ex vivo in patients enrolled on a single agent phase II trial of ibrutinib in CLL (NCT01500733) and investigated the mechanism.In order to evaluate the effect of ibrutinib on CLL cell adhesion in vivo we compared peripheral blood tumor cells collected from patients pre-treatment and on day 28 of treatment. Fluorescently labeled cells were plated on fibronectin-coated plates and allowed to adhere for 1 hour. Adhesion was then evaluated using florescence microscopy and quantified using the ImageJ software. After 28 days on ibrutinib, CLL cells displayed a severely reduced adhesion ability (99% reduction, P<0.001, n=13). Next, we tested the effect of ibrutinib on CLL cell adhesion to a confluent stromal cell layer. Although less dramatic, we found a similar reduction in the ability of cells to adhere to the stromal cell layer in all three patients evaluated. As the initial rise in ALC occurs very rapidly in some patients, we evaluated the ability of cells collected on day 2 of ibrutinib therapy and again found a significant inhibition of cell adhesion to fibronectin (81% reduction, P=0.005, n=10). This suggests that even one dose of ibrutinib is sufficient to disrupt CLL cell adhesion.We next sought to determine if ibrutinib could directly interfere with adhesion by causing cells to detach from fibronectin. We found that after one hour of 1µM ibrutinib treatment in vitro there was a significant reduction in the number of CLL cells that remained attached (33% reduction, P=0.03, n=10). This suggests that ibrutinib can not only prevent CLL cell adhesion but can release adhered cells from the microenvironment, consistent with influx of cells from the lymph node into the peripheral blood within hours of starting treatment.To determine whether the observed defect in cell adhesion is due to changes in the expression of CD29/CD49d (VLA-4 or α4β1 integrin), or CD18/CD11a (LFA-1), we measured the expression of these known regulators of CLL cell adhesion in samples obtained pre-treatment and during treatment. While there was inter-patient variability, overall there was no significant change in expression of CD29 after 28 days on treatment. In contrast albeit minor, we found reductions in both CD49d and CD18 (P<.05). When analyzing cells obtained on day 2, we found no significant changes suggesting that alteration of adhesion molecule expression does not account for the rapid increase in ALC. Integrin α4β1 is activated by PKC downstream of BTK. We thus sought to determine whether stimulation with PMA, an activator of PKC, could restore the ability of CLL cells to adhere to fibronectin. Indeed, PMA stimulation of CLL cells obtained from patients after 28 days on ibrutinib partially restored their ability to adhere to fibronectin, suggesting that reduced α4β1 integrin activity contributes to inhibition of adhesion by ibrutinib.In conclusion, ibrutinib abolished CLL cell adhesion at least in part due to direct inhibition of BTK- and BCR-mediated α4β1 integrin activity. Additionally, ibrutinib not only prevented adhesion but also caused the detachment of adhered cells from the extracellular matrix. Together, this suggests that disruption of cellular adhesion is a major contributor to the observed rise in ALC early on treatment.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood samples to make this research possible. DisclosuresWiestner:Pharmacyclics Inc.: Research Funding.

CD40-Mediated Activation of Chronic Lymphocytic Leukemia Cells Promotes Their CD44-Dependent Adhesion to Hyaluronan and Restricts CCL21-Induced Motility

Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes, provide signals needed for activation and outgrowth of the tumor clone. Furthermore, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration, and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral lymph nodes, focusing on its regulation via CD40L-dependent, T-cell-mediated activation of CLL cells. We found that hyaluronan triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L stimulation promoted the firm, CD44-mediated adhesion of CLL cells to hyaluronan, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L activation, seemed to facilitate hyaluronan recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within lymph node compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T-cell stimuli and facilitate essential interactions with hyaluronan-bearing stromal cells, collectively promoting CLL cell proliferation and survival.

The Elastin Microfibril Interfacer-1 (EMILIN-1) Is a Ligand for CD49d in Chronic Lymphocytic Leukemia Cells

AbstractAbstract 1772CD49d (α4 integrin chain) is a strong negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell microenvironmental interactions. CD49d triggering by its main ligands Vascular Cell Adhesion Molecule-1 (VCAM-1) and non-RGD sites (CS-1 fragments) of fibronectin (FN) activates signalling pathways delivering pro-survival signals, and promoting resistance to drug-induced apoptosis in CLL. Recently, the globular (g) C1q-like domain of Elastin MIcrofibriL INterfacer1 (EMILIN1), an adhesive extracellular matrix constituent, was described as a new ligand for CD49d, where it operates as a negative modulator of proliferation signals in substrate-adherent non-hematopoietic CD49d+ cells (Danussi et al, J Cell Biol, 2011). Here, we investigated the distribution of EMILIN1 in normal and CLL-involved tissues, and the effects of EMILIN1/CD49d interaction in CLL in terms of adhesion and survival.By taking advantage of a specific anti-human EMILIN1 monoclonal antibody (Spessotto et al, J Biol Chem, 2003), exploratory staining in reactive lymphoid tissues (tonsil) indicated a clear extracellular EMILIN1 specific reactivity in the outer zone of the mantle/marginal areas. When investigated in lymph node tissues from CLL cases (n=3) by both immunohistochemical and immunofluorescence analyses, a clear EMILIN1 positive staining was detected intermingled with the neoplastic component. To verify whether EMILIN1 could promote CLL cell adhesion, we took advantage of the CLL-derived CD49d+ Mec-1 CLL-like cell model, previously demonstrated by us to adhere to both VCAM-1 and FN (Zucchetto et al, Leukemia, 2012). Results demonstrated similar adhesion levels of Mec-1 cells on VCAM-1, CS-1 fragment of FN and the gC1q-like EMILIN1 domain (mean number of adherent cells per field 267±24, 272±7 and 317±21, respectively).Consistently, adhesion experiments with primary CLL cells characterized by high and homogeneous CD49d expression confirmed similar levels of adhesion on both VCAM-1 and EMILIN1 (mean number of adherent cells per field 141±55 and 135±76, respectively). In all cases adhesion was specifically blocked by pre-treatment with the anti-CD49d HP1/2 blocking antibody. Immunofluorescence analysis with Mec-1 and primary CLL cells showed the recruitment of phospho-Vav-1 at the CD49d/EMILIN1 adhesion sites, with concomitant F-actin reorganization, confirming the activation of the integrin signalling pathway.The effects of EMILIN1/CD49d interactions in CLL were next investigated performing short-term adhesion experiments (2 and 5 minutes) on gC1q-like EMILIN1 domain and VCAM-1 using CLL cells from three CD49d+ cases, and verifying the phosphorylation of Akt and ERK1/2 kinases, key mediators of survival signals by western blotting. In all cases, an increased intensity of Akt and ERK1/2 phosphorylation was documented after two-minutes of adhesion on both EMILIN1 (mean fold increase =1.2±0.1, and =5.0±1.0 as compared to controls, respectively) and VCAM-1 (mean fold increase =1.6±0.2, and =4.0±1.9, respectively). The phosphorylation was even greater after five minutes (mean fold increase =2.4±0.5, and =10.0±4.0, for EMILIN1 and =2.4±0.6 and =5.0±0.7 for VCAM-1, respectively). The concomitant increase of phospho-Vav-1 confirmed the activation of the integrin signalling pathway.Finally, we verified whether CD49d/EMILIN1 interaction was able to protect CLL cells from spontaneous apoptosis, by culturing purified cells from 6 CLL cases on gC1q-like EMILIN1 domain, VCAM-1, or control substrate (1% BSA), and checking cell viability after 5 days. Both VCAM-1 and EMILIN1 were able to protect CLL cells from spontaneous apoptosis (p=0.03 and p=0.001, respectively), the viability obtained on EMILIN1 being also significantly higher than that observed on VCAM-1 (p=0.007).In conclusion, we showed here for the first time that EMILIN1 is present in normal and CLL-involved lymphoid tissues, and it is able to efficiently bind to CD49d, as expressed by CLL cells. At variance of what demonstrated in non-hematopoietic models, EMILIN1 was shown to be able to deliver anti-apoptotic/pro-survival signals to circulating CLL cells. EMILIN1/CD49d interactions may have a role in the maintenance of the neoplastic clone in CD49d-expressing CLL. Disclosures:No relevant conflicts of interest to declare.

Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction

AbstractAbstract 3900In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche.This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures:No relevant conflicts of interest to declare.

The Bruton's tyrosine kinase (BTK) inhibitor PCI-32765 (P) in treatment-naive (TN) chronic lymphocytic leukemia (CLL) patients (pts): Interim results of a phase Ib/II study.

6507 Background: Fludarabine based therapy, while effective, carries significant risk of morbidity and mortality in the elderly pts. As such, older CLL pts represent a high priority for new therapeutic approaches. PCI-32765 (P) an oral, selective, irreversible inhibitor of BTK inhibits CLL cell proliferation, migration and adhesion. A multi-cohort Phase Ib/II trial evaluated 2 doses of single-agent P in both TN and relapsed/ refractory (R/R) CLL/SLL pts. Mature follow-up of the TN pts is reported. Methods: Pts &gt;65 yrs old with active CLL requiring Tx by IWCLL guidelines were treated with oral P at doses of 420 mg or 840 mg administered daily for 28-day cycles until disease progression (PD). Response was evaluated according to 2008 IWCLL criteria. Results: 31 pts were enrolled- 26 pts (420mg) and 5 pts (840 mg). The 840 mg cohort was terminated after comparable activity and safety between doses was shown in R/R pts. Median age 71 yrs (range 65-84) with 74% of pts &gt;70 yrs. 19/31 (61%) had baseline cytopenias (Hgb &lt; 11g/dl or plts &lt;100,000). Unmutated IgVH was present in 43% of pts. The majority of AEs have been Gr&lt;=2 in severity, most commonly diarrhea, nausea, and fatigue. Gr &gt;3 non-heme AEs potentially related to P in 19% of pts. 10% of pts experienced Gr &gt;3 infections or cytopenias. With a median follow-up of 10.7 mos on 420 mg cohort 73% (19/26) achieved a response by IWCLL criteria with 65% partial responses (PR) and 8% complete remissions with no morphologic evidence of CLL on marrow. An additional 12% (3/26) of pts achieved nodal responses (NR) with lymphocytosis. Median follow-up in the 840 mg cohort is 4.6 mo, at 2 cycle assessment 2/5 achieved a PR and 1 pt with a NR. ORR was independent of high risk factors. 84% of pts remain on study, reasons for discontinuation = AE (3), investigator decision (1) and PD (1). There have been no deaths. Estimated 12 mo median PFS for the 420 mg cohort is 93%. Conclusions: PCI-32765 is highly active and well tolerated in elderly TN CLL pts. The high ORR, including marrow clearance and very low PD rate with the single agent suggests that P warrants further study as a first-line treatment approach in elderly pts.