CLL-derived Cell Lines Research Articles

Aberrant BCAT1 expression augments MTOR activity and accelerates disease progression in chronic lymphocytic leukemia.

We performed gene expression profiling of mRNA/cDNA isolated from N = 117 flow sorted CLL. We detected aberrant expression of the metabolic enzyme branched chain amino acid transferase (BCAT1) in CLL with del17p/TP53mut. Through extensive validation, we confirmed the highly preferential expression of BCAT1 in CLL with del17p/TP53mut (66%) or trisomy 12 (77%). BCAT1 was not expressed in B cells isolated from normal human lymph nodes. The products of the bidirectional BCAT1 reaction, including leucine, acetyl-CoA, and alpha-ketoglutarate are known activators of MTOR. We measured an ~two-fold higher MTOR activity via normalized p-S6K levels in primary CLL with BCAT1 high versus absent expression before and after sIgM crosslinking. Through steady state metabolomics and heavy isotope metabolic tracing in primary CLL cells, we demonstrate that CLL cells are avid consumers of branched chain amino acids (BCAAs) and that BCAT1 in CLL engages in bidirectional substrate reactions. Of additional interest, CLL with aberrant BCAT1 expression were less sensitive to Venetoclax-induced apoptosis. Biologically, three CLL-derived cell lines with disruption of BCAT1 had substantially reduced growth ex vivo. Clinically, the expression of any detectable BCAT1 protein in CLL independently associated with shorter median survival (125 months versus 296 months; p < 0.0001), even after exclusion of del17p/TP53mut cases.

P66Shc deficiency in CLL cells enhances PD-L1 expression and suppresses immune synapse formation.

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eμ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

TRAF3 Alterations Identify a Subgroup of Patients with Adverse Clinical Outcome and Induce Metabolic Reprogramming in Chronic Lymphocytic Leukemia

The TNF receptor-associated factor3 (TRAF3) gene located in 14q32 is frequently altered by deletion and/or mutation in multiple myeloma, lymphoma and other B-cell malignancies such as chronic lymphocytic leukemia (CLL). TRAF3 is a negative regulator of the non-canonical NF-KB signaling, and recent data uncovered additional functions of this gene in inflammation, antiviral immunity or cellular metabolism. Nevertheless, the biological and clinical significance of TRAF3 alterations in CLL is unknown. For that, the aim of this study was to assess the clinical impact of TRAF3 alterations in a CLL cohort as well as its biological role in CLL pathogenesis through CRISPR/Cas9-generated models. TRAF3 alterations (mutations and copy number variations-CNVs) were assessed by NGS in our previously published cohort of 340 CLLs (Pérez Carretero, AJH 2022). A total of 14 patients exhibited a TRAF3 CNV loss, and 10 of them showed additional TRAF3 loss-of-function mutations, indicating a biallelic inactivation of this gene in CLL. Interestingly, the presence of TRAF3 alterations was significantly associated with shorter time to first treatment (6 vs 75 months, p<0.001), being an independent risk factor in the multivariate analysis (p=0.029). To further investigate the biological role of TRAF3, we used the CRISPR/Cas9 system to model TRAF3 loss-of-function mutations in the CLL-derived cell line PGA1, and characterized these cells by transcriptomics, metabolomics and functional studies. RNA-seq analyses identified 54 dysregulated genes enriched in pathways such as NIK/NF-kB signaling (FDR=0.029). Concretely, NIK (a direct target of TRAF3-mediated degradation) was significantly upregulated (Q<0.001) in TRAF3mut clones, which correlated with a higher nuclear activity of the non-canonical NF-kB transcription factors p52 (p=0.05) and RelB (p=0.02). Moreover, we validated these results in primary CLL cells (n=15; 3 TRAF3mut cells). Besides, PGA1-TRAF3mut cells showed increased levels of BAX (p=0.05) and more sensitivity to venetoclax (IC50 3.1 vs 13.8μM, p=0.003). In the metabolomic study, we found an enrichment of metabolites involved in Warburg effect, citric acid cycle (TCA) and glutamate metabolism (FDR<0.001, 0.003, 0.1 resp.), with significant higher levels of α-ketoglutarate (TCA intermediate synthesized from glutamate), pyruvate and lactate (products of glycolysis) (p<0.001, p<0.001, p=0.002) in PGA1-TRAF3mut cells. To better analyze their metabolic specificities, we evaluated the mitochondrial oxygen consumption rate (OCR) extracellular acidification (ECAR) and cell viability at basal state and in response to metabolic inhibitors. Of note, PGA1-TRAF3mut cells exhibited distinct mitochondrial characteristics: higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis and glycolytic reserve were also enhanced in the TRAF3mut cells. In this way, they were more sensitive to 2-deoxy-D-glucose (glucose uptake inhibitor), and more resistant to oxamate (lactate-dehydrogenase inhibitor, marker of anaerobic glycolysis). Overall, these results indicate, unlike WT, TRAF3mut cells instead utilize a mitochondrial glycolytic metabolism. Surprisingly, by using UK5099 (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) mutant cells showed higher maximal respiration levels than WT, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. We therefore tested the effect of C968 (glutaminolysis inhibitor) and we found that TRAF3mut cells were also more resistant to this drug, what reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis) in the PGA1-TRAF3mut cells that does not exist in the WT. Interestingly, the combined treatment (UK5099 and C968) induced more cell death in the TRAF3mut than in the WT cells (p=0.03), suggesting a new potential opportunity to treat patients with TRAF3 alterations. In conclusion, TRAF3 deficiency appeared to be an independent prognostic factor of time to first treatment in CLL. This alteration results in a non-canonical NF-KB activation and in a metabolic reprogramming towards an enhanced mitochondrial glycolysis and glutaminolysis, which could be potentially targetable in CLL patients. Fund.: PI21/00983 FI19/00191 FEHH

NOTCH1 Signaling Is Dysregulated in CLL By the Deubiquitinase USP28 That Is Recurrently Affected By Del(11q) and Can be Therapeutically Targeted

Active NOTCH1 signaling is present in approximately half of chronic lymphocytic leukemia (CLL) patients and was shown to be a marker for disease outcome. The NOTCH1 signaling pathway can be aberrantly activated through mutations in the NOTCH1 gene itself or co-factors such as FBXW7, resulting in protein stabilization of cleaved NOTCH1 (NICD). However, 50% of CLL cases with NOTCH1 activation do not harbor alterations in these genes, suggesting additional mechanisms for NOTCH1 hyperactivation. Since NICD can be stabilized due to defects in the ubiquitination/degradation machinery, we aimed to elucidate the role of the on chromosome 11q23 located deubiquitinase USP28 in the regulation of NOTCH1 activity and as a novel therapeutic target for NOTCH1-dependent CLL patients. First, we evaluated USP28 expression levels in a previously published cohort of 285 untreated CLL patients characterized via SNP array analysis and gene expression profiling (Edelmann et al, Blood 2012; Bloehdorn et al, Nat Commun 2021). The presence of monoallelic USP28 deletion was identified in 90% of CLL patients harboring a heterozygous deletion of chromosome 11q (del(11q); n=96), resulting in a significant decrease of USP28 RNA expression in comparison to non-del(11q) cases (n=199, P<0.0001). In non-del(11q) cases, USP28 expression levels were variable and assessment of protein levels of USP28 and its target proteins by western blotting in primary CLL cells (n=8) revealed a correlation between high protein levels of USP28 and remarkably high levels of NICD and other USP28 targets such as c-MYC or c-JUN. In addition, interaction of USP28 with NICD was shown by immunoprecipitation assays. In parallel, we implemented the CRISPR/Cas9 system to generate HG3 CLL-derived cell lines harboring USP28 biallelic deletion (HG3-USP28KO, n=5), monoallelic 11q deletion (HG3-del(11q), 17 Mb size including both ATM and USP28 genes, n=3), biallelic ATM truncation (HG3-ATMKO, n=3) or the combination of both events (HG3-del(11q) ATMKO, n=3). Overexpression of USP28 in HG3WT and HG3-USP28KO cells increased NOTCH1 activity measured by luciferase reporter assays (P<0.05, P<0.001, respectively). In contrast, in HG3-del(11q) cells decreased USP28 RNA and protein expression resulted in reduced NOTCH1 activity compared to HG3WT cells (P<0.05). Notably, HG3-del(11q) ATMKO cell lines showed even lower levels of NOTCH1 activity (P<0.01). Given that ATM phosphorylates USP28 (Zhang et al, Cell 2006), we hypothesized that in CLL cells ATM regulates USP28 activity and thereby also downstream NOTCH1 signaling. Indeed, NOTCH1 activity was reduced in HG3-ATMKO cells (P<0.01). However, USP28 overexpression in HG3-ATMKO cells did not lead to increased NOTCH1 signaling activity as observed in HG3WT cells. Moreover, pharmacological ATM inhibition (5 µM of KU-55933) also resulted in reduced NOTCH1 activity in HG3WT cells (P<0.0001) and reduced NICD levels in primary CLL cells (n=12; P<0.05). We finally explored the impact of the small molecule USP28 inhibitor AZ1 (alone or in combination with ibrutinib or venetoclax) on NICD protein levels and viability of primary CLL cells (n=15). AZ1 treatment (10 µM) resulted in decreased NICD levels, independent of mutations within NOTCH1 or FBXW7 (P<0.01). ATP quantification and 7-AAD staining revealed that increasing doses of AZ1 significantly reduced viability of NOTCH1/FBXW7-MUT primary cells (n=5) as well as of NOTCH1/FBXW7-WT cells (n=6) expressing high protein levels of USP28 and NICD (P<0.01; P<0.05, respectively). In contrast, AZ1 treatment did not affect viability of CLL patient cells expressing low NICD levels (n=4; P=0.56) Furthermore, AZ1 combined with venetoclax potentiated the reduction of cell viability especially in NOTCH1/FBXW7-MUT or patient cells expressing high USP28 and NICD levels (P<0.001; P<0.05). We propose that high levels of the deubiquitinase USP28 are responsible for hyperactivation of NOTCH1 signaling in CLL. This hypothesis is further confirmed in del(11q) CLL cells, where USP28 heterozygous deletion resulted in low NOTCH1 activity. In addition, our data reveal that USP28 activity is regulated by ATM, uncovering a novel mechanism of NOTCH1 modulation via the ATM-USP28-NOTCH1 axis (Figure 1). Finally, we demonstrate that USP28 inhibition by AZ1 suppresses USP28-mediated NOTCH1 activation and could represent a novel therapeutic strategy in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P66Shc Deficiency in Chronic Lymphocytic Leukemia Promotes Chemokine Receptor Expression Through the ROS-Dependent Inhibition of NF-κB.

The microenvironment of lymphoid organs is central to the pathogenesis of chronic lymphocytic leukemia (CLL). Within it, tumor cells find a favourable niche to escape immunosurveillance and acquire pro-survival signals. We have previously reported that a CLL-associated defect in the expression of the pro-apoptotic and pro-oxidant adaptor p66Shc leads to enhanced homing to and accumulation of leukemic cells in the lymphoid microenvironment. The p66Shc deficiency-related impairment in intracellular reactive oxygen species (ROS) production in CLL cells is causally associated to the enhanced expression of the chemokine receptors CCR2, CXCR3 and CCR7, that promote leukemic cell homing to both lymphoid and non-lymphoid organs, suggesting the implication of a ROS-modulated transcription factor(s). Here we show that the activity of the ROS-responsive p65 subunit of the transcription factor NF-κB was hampered in the CLL-derived cell line MEC-1 expressing a NF-κB-luciferase reporter following treatment with H2O2. Similar results were obtained when intracellular ROS were generated by expression of p66Shc, but not of a ROS-defective mutant, in MEC-1 cells. NF-κB activation was associated with increased expression of the chemokine receptors CCR2, CXCR3 and CCR7. Reconstitution of p66Shc in CLL cells normalized intracellular ROS and hampered NF-κB activation, which led to a decrease in the expression of these homing receptors. Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to NF-κB hyperactivation and homing receptor overexpression, providing a mechanistic basis for the enhanced ability of these cells to accumulate in the pro-survival lymphoid niche.

Idelalisib activates AKT via increased recruitment of PI3Kδ/PI3Kβ to BCR signalosome while reducing PDK1 in post-therapy CLL cells.

Idelalisib targets PI3Kδ in the BCR pathway generating only a partial response in CLL patients, indicating that the leukemic cells may have evolved escape signals. Indeed, we detected increased activation of AKT accompanied by upregulation of MYC/BCL2 in post-therapy CLL cells from patients treated with idelalisib/ofatumumab. To unravel the mechanism of increased AKT-activation, we studied the impact of idelalisib on a CLL-derived cell line, MEC1, as a model. After an initial inhibition, AKT-activation level was restored in idelalisib-treated MEC1 cells in a time-dependent manner. As BCAP (B-cell adaptor for PI3K) and CD19 recruit PI3Kδ to activate AKT upon BCR-stimulation, we examined if idelalisib-treatment altered PI3Kδ-recruitment. Immunoprecipitation of BCAP/CD19 from idelalisib-treated MEC1 cells showed increased recruitment of PI3Kδ in association with PI3Kβ, but not PI3Kα or PI3Kγ and that, targeting both PI3Kδ with PI3Kβ inhibited AKT-reactivation. We detected similar, patient-specific recruitment pattern of PI3K-isoforms by BCAP/CD19 in post-idelalisib CLL cells with increased AKT-activation. Interestingly, a stronger inhibitory effect of idelalisib on P-AKT (T308) than S473 was discernible in idelalisib-treated cells despite increased recruitment of PI3Kδ/PI3Kβ and accumulation of phosphatidylinositol-3,4,5-triphosphate; which could be attributed to reduced PDK1 activity. Thus, administration of isoform-specific inhibitors may prove more effective strategy for treating CLL patients.

Clinical and Biological Impact of TP53 Alterations in Del(11q) Chronic Lymphocytic Leukemia

Large-scale next-generation sequencing (NGS) studies have suggested common patterns of co-occurrence or mutual exclusivity between genetic alterations in chronic lymphocytic leukemia (CLL). However, little is known about how most of these alterations cooperate to drive CLL pathogenesis, as well as the impact of these concurrencies in clinical outcome. In this regard, we investigated the clinical and biological impact of the co-occurrence of high-risk lesions such as del(11q)/ATM mutation and del(17p)/TP53 mutation by integrating NGS and CRISPR/Cas9 approaches. To address these questions, we first analyzed the mutational profile of 271 CLLs (17.3% del(11q); 10.7% del(17p)). The most frequently mutated genes were NOTCH1 (20%), TP53 (14%), SF3B1 (11%) and ATM (10%). Within del(11q), 32% showed TP53 alterations (53% biallelic; 47% monoallelic). Interestingly, patients harboring combined del(11q) and TP53 alterations by either mutation or deletion (del(11q) TP53ALT) exhibited significantly shorter overall survival (OS) than del(11q) CLLs without TP53 alterations (del(11q) TP53WT) and those TP53 altered without del(11q) (no del(11q) TP53ALT) (median 17 vs. 88, 36 months; P=0.0004, P=0.02). Conversely, we observed a significant lack of ATM mutations in CLLs with biallelic TP53 alterations (P=0.002) and a mutual exclusivity between biallelic TP53 and biallelic ATM losses (P=0.03)(Fig 1A). Based on the NGS results, we next used the CRISPR/Cas9 system to model monoallelic and biallelic ATM and TP53 loss in vitro. We generated isogenic HG3-Cas9 CLL-derived cell lines harboring monoallelic del(11q) (targeting 11q22.1/11q23.3 regions) and further loss-of-function mutations in ATM and/or TP53 to mimic all the possible combinations observed in our CLL cohort. By proliferation assays, we noted that the introduction of TP53 mutations increased the proliferation rates in both HG3WT and HG3-del(11q) cells. In contrast, the introduction of an ATM truncating mutation on the remaining allele of the HG3-del(11q) TP53MUT clone, suppressed this proliferative advantage, with growth rates comparable to those of HG3-del(11q). Accordingly, DNA content analysis by propidium iodide revealed that cells harboring biallelic ATM and TP53 loss also showed mitotic and cell cycle defects. To further evaluate the implications of these alterations in the clonal dynamics of CLL in vivo, we performed fluorescence-based clonal competition experiments by injecting these edited cell lines intravenously into NGS mice. First, we observed that HG3-TP53MUT cells outgrew HG3WT cells in spleen of xenotransplanted mice 14 days after injection (P&amp;lt;0.001). In a second experiment, HG3-del(11q), HG3-del(11q) TP53MUT and HG3-del(11q) ATMMUTTP53MUT cells were injected. Strikingly, HG3-del(11q) TP53MUT cells were able to outcompete HG3-del(11q) cells in spleen and bone marrow (P&amp;lt;0.001; P&amp;lt;0.001). By contrast, HG3-del(11q) ATMMUTTP53MUT cells failed to engraft neither in spleen nor bone marrow, being outcompeted by the other injected cells (P&amp;lt;0.001), providing biological insights on the mutual exclusivity of these genetic events in CLL (Fig 1B). We next assessed whether these cell models could predict responses of these combined abnormalities to BTK and PI3K inhibitors. We observed that HG3-del(11q) TP53MUT and HG3-TP53MUT cells showed partial response to ibrutinib and idelalisib, although the IC50 values were still higher than the ones observed in HG3WT clones, especially with idelalisib (27.4 and 20.6 vs. 1.8 uM, respectively). Nonetheless, we found that HG3-del(11q) TP53MUT cells were highly sensitive to novel preclinical drugs that have been shown to be effective in TP53 deficient cells such ATR inhibitors, with an IC50 value comparable to HG3WT cells (mean IC50 0.55 vs. 0.67 uM) (Fig 1C). In summary, we show that mutations in TP53 can appear in a subset of monoallelic del(11q) CLL cases, conferring a synergistic clonal advantage in vivo, and therefore a dismal clinical impact on the OS of this CLL subgroup. In addition, the biological basis of mutual exclusivity of biallelic ATM and TP53 alterations in CLL was assessed, underscoring the importance of the number of alleles affected by these alterations, and establishing novel pre-clinical models for the study of the biology and therapeutic response of concurrent genetic abnormalities in the disease. Funding: PI18/01500 FI19/00191 CD19/00222 *MQA CPC equal contr Disclosures No relevant conflicts of interest to declare.

Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

BackgroundWhile achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence...

Suppression of minichromosome maintenance 7 expression sensitizes chronic lymphocytic leukemia cells to fludarabine

Chronic lymphocytic leukemia (CLL) constitutes the largest percentage of adult leukemia cases in Western countries. Classically, fludarabine (Flu) is an effective drug used as a first-line therapy for CLL; however, Flu resistance limits its clinical effect. Minichromosome maintenance (MCM) complex components 2–7 exert important functions in maintaining genomic stability. Replication stress occurs upon dysregulation of MCM7, which potentiates malignant phenotypes. In this study, primary CLL cells and CLL-derived cell lines displayed elevated MCM7 expression. In CD40-stimulated primary CLL cells, MCM7 inhibition resulted in increased Flu-induced apoptosis and delayed repair of DNA damage. In the MEC-1 and EHEB cell lines, knockdown of MCM7 with lentivirus significantly inhibited cell proliferation and promoted cell cycle arrest at S phase. Moreover, MCM7 silencing sensitized both cell lines to Flu by increasing replication stress. The combination of Flu administration with MCM7 inhibition represents a novel approach to reverse Flu resistance in CLL.

CC-122 is more potent but transcriptionally mirrors Lenalidomide in Chronic Lymphocytic Leukemia

Abstract Lenalidomide (Len) is an immune modulatory drug for the treatment of hematological malignancies which contrasts with a large repertoire of immunosuppressive treatments. Len is FDA approved for Multiple Myeloma (MM), yet its development in Chronic Lymphocytic Leukemia (CLL) has been hampered by a potentially fatal, dose-limiting toxicity known as tumor flare. Tumor flare consists of enlarged, painful lymph nodes (LN) and cytokine release and is managed by lower dosage/prophylactic measures. Len and next-generation reagent CC-122, target cereblon, leading to degradation of transcription factors IKZF1/IKZF3. We compared CC-122 to Len using primary CLL B and T cells and CLL-derived cell line, OSU-CLL. We present a key difference between CC-122 and Len treated activated CLL T cells. CC-122 0.1–10uM or 1uM Len are not directly cytotoxic to primary CLL B cells (avg. fold change 0.94–1.49 normalized to vehicle, N=8). CC-122 was more potent than Len by degradation of IKZF1/IKZF3, and immune activation measured by CD86 induction (7.2% more CD86+ induction, p=0.0026, N=12). RNA-seq analysis of the OSU-CLL cell line showed that at physiologically relevant doses, CC-122 (0.1uM) and Len (1uM) have no significant differences. Microarray analysis of 0.1uM CC-122 v. 1uM Len on αCD3 activated CLL T cells showed only two significantly different genes: CXCL13 and HLADQ-A1, p&amp;lt;0.0001. CXCL13, a B cell chemoattractant in CLL, was ≥ 3 fold increased with 1uM Len v. 0.1–1uM CC-122 (N=3). Overexpression of the CXCL13 receptor, CXCR5, in CLL but not MM may contribute to migration of CLL cells to the LN and potentially tumor flare. Retaining the anti-tumor activity of Len while mitigating tumor flare is of clinical interest and critical to the development of CC-122.

Abstract LB-289: Interaction between miR-155 and Quaking in the innate immune response and leukemia

Abstract Rationale: miR-155 is a pro-inflammatory microRNA upregulated in human and mouse macrophages exposed to lipopolysaccharide (LPS) that is required to mount an effective immune response. High levels of miR-155 are observed in different solid tumors as well as leukemias, including chronic lymphocytic leukemia (CLL). Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein. In silico analyses predict that the QKI transcript is a target of miR-155, and we hypothesized that miR-155 might carry out its pro-inflammatory and oncogenic signals at least in part by targeting QKI. Methods: Mouse RAW-264.7 macrophages were stimulated with LPS or mock PBS three times over a period of 6 days. qRT-PCR was used to monitor the expression of QKI, miR-155 and Tnf (tumor necrosis factor alpha), and Quaking protein (Qki) expression was analyzed by Western blot. Expression of miR-155 and QKI in Burkitt's lymphoma cell lines and CLL cell lines was measured by qRT-PCR; QKI expression was also determined by Western blot. Cell lines were transfected with miR-155 or miR-control, or 155-I, and Western blot analysis of QKI was performed after 48 hours. To study the in vivo effects of the cooperation between miR-155 and QKI, we analyzed Qki expression in splenic B cells from wild type C57B1/6 mice, miR-155−/- mice, and Eμ-miR-155 transgenic mice (which develop aggressive CLL), with and without LPS challenge. Finally, expression of Qki was determined in samples from patients with B-cell CLL. Results: After 8 hours of LPS challenge there was a 2-fold decrease in QKI expression, while miR-155 and Tnf both increased approximately 10-fold (p&amp;lt;0.05). QKI returned to pre-treatment levels at 2 days, while miR-155remained high for 3 days. LPS re-stimulation at 3 days (mimicking chronic inflammation) reduced QKI expression 2.3-fold (p&amp;lt;0.05) over a 48 hour period in parallel with a renewed up-regulation of miR-155, and Western blotting confirmed the above observed changes for Qki. CLL-derived cell lines showed significantly higher expression of miR-155, and lower expression of QKI, compared with Burkitt's lymphoma cell lines (p&amp;lt;0.05). Transfection of miR-155 led to decreased Qki expression in Burkitt's lymphoma lines, while 155-I transfection of MEC2 CLL cells caused Qki upregulation at 48 hours. Splenic B cells from leukemic Eμ-miR-155 mice had decreased Qki levels compared with C57B1/6 mice and miR-155−/−mice, and LPS challenge of cultured B cells resulted in a pronounced downregulation of Qki expression only in B cells coming from Eμ-miR-155 mice. Finally, reduced expression of Qki was observed in B cells coming from patients with CLL compared with healthy patients. Conclusions: The inverse relationship between miR-155 and Qki is specific, and may represent a regulatory mechanism for the immune response mounted by macrophages and B cells. This relationship also sheds light on the pro-inflammatory and oncogenic properties of miR-155 in leukemia. Citation Format: Timothy K. Richmond, Esmerina Tili, Melissa Brown, Marcela Chiabai, Dario Palmieri, Ri Cui, Tyler Sheetz, Carlo M. Croce. Interaction between miR-155 and Quaking in the innate immune response and leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2015-LB-289

OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells

AbstractAberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL.

High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle.

Myc (c-Myc) counteracts p27 effects, and low p27 usually correlates with high Myc expression in human cancer. However there is no information on the co-expression of both genes in chronic lymphocytic leukemia (CLL). We found a lack of correlation between RNA and protein levels of p27 and Myc in CLL cells, so we determined the protein levels by immunoblot in 107 cases of CLL. We observed a high p27 protein expression in CLL compared to normal B cells. Ectopic p27 expression in a CLL-derived cell line resulted in cell death resistance. Surprisingly, Myc expression was very low or undetectable in most CLL cases analyzed, with a clear correlation between high p27 and low Myc protein levels. This was associated with low Skp2 expression, which is consistent with the Skp2 role in p27 degradation and with SKP2 being a Myc target gene. High Myc expression did not correlate with leukemia progression, despite that cell cycle-related Myc target genes were upregulated. However, biochemical analysis showed that the high p27 levels inhibited cyclin-Cdk complexes even in Myc expressing CLL cells. Our data suggest that the combination of high p27 and low Myc is a marker of CLL cells which is mediated by Skp2.

OSU-T315, An Integrin-Linked Kinase (ILK) Inhibitor, Induces Apoptosis By Targeting B Cell Receptor and CD49d Mediated AKT/ERK Activation In Chronic Lymphocytic Leukemia Cells

Aberrant regulation of endogenous survival pathways plays a major role in pathogenesis of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche is believed to activate survival and proliferation pathway in CLL. PI3K/AKT appears to be the pivotal axis to support CLL growth. Current studies identify Integrin-linked kinase (ILK) as a Phosphoinositide-dependent kinase-2 (PDK2), by which it mediates AKT activation and concomitant with tumorigenesis in many solid tumors. As a result of our search for novel therapeutic targets, we first report the potent inhibitor, OSU-T315, modeled from the scaffold that docks into AKT-binding site of ILK in therapeutic of hematological malignancy. OSU-T315 triggers apoptosis in CLL by targeting both intrinsic and extrinsic AKT and ERK activation (Figure 1A). OSU-T315 induces cytotoxicity to both CLL-derived cell lines Mec-1 and OSU-CLL, as well as in primary CLL cells, while illustrating alleviated toxicity in normal B and T cells (Figure 1B, C). In contrast to the highly successful BTK and PI3 kinase inhibitors which inhibit BCR signaling proximally, OSU-T315 specifically inhibits down-stream AKT and ERK signaling cascades without affecting proximal BCR or focal-adhesion signaling. In particular, external stimuli through BCR or CD49d, by which triggers survival signal and protects CLL from ankoisis via AKT/ERK activation, is potently inhibited by OSU-T315. Downstream anti-apoptotic molecule Mcl-1 is suppressed upon OSU-T315 treatment, which is later accompanied by increased Caspase 3/7 activity. Further study indicates that OSU-T315 mediated cell death is Caspase dependent. In vivo efficacy of OSU-T315 is supported by a therapeutic study in the TCL1 mouse model of CLL, from which demonstrates improvement in total counts of white blood cells and reduced splenomegaly, thereby contributing to enhanced overall survival following OSU-T315 oral treatment regimen (Figure 2). Together, our findings suggest the innovative dual-targeted approaches by suppressing both AKT and ERK pathway in CLL therapy through inhibiting ILK, and provide an alternative agent for potential therapeutic development in CLL. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Molecular characterization of neoplastic and normal “sister” lymphoblastoid B-cell lines from chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.

Real-World Efficacy and Safety of Bendamustine with or without Rituximab in Treatment-Naïve Patients with Chronic Lymphocytic Leukemia: Retrospective Analysis of a German Registry.

AbstractAbstract 2905 BackgroundBendamustine, a unique alkylating agent with a multifaceted mechanism of action is effective front-line therapy for chronic lymphocytic leukemia (CLL). In vitro studies showing that cytotoxic activity of bendamustine against CLL-derived cell lines is synergized by rituximab, an anti-CD20 monoclonal antibody, have led to investigation of combination bendamustine plus rituximab (BR) as first-line therapy and treatment of relapsed disease. This retrospective analysis assessed real-world efficacy and safety of bendamustine alone and combined with rituximab in treatment-naïve CLL patients from Projektgruppe Internistische Onkologie, the largest registry of treatment data from private medical oncology practices in Germany. MethodsRecords were obtained for all CLL patients in a registry from 57 German oncology practices from May 2008 to July 2011. Patients who received ≥3 cycles of first-line bendamustine monotherapy or BR were divided into the following age/treatment groups: ≤60 years treated with BR ± prednisone (P) [≤60 BR]; >60 to <70 years treated with bendamustine monotherapy [60–70 B]; >60 to <70 years treated with BR ± P [60–70 BR]; ≥70 years treated with bendamustine monotherapy [≥70 B]; and ≥70 years treated with BR ± P [≥70 BR].The primary efficacy measure was ORR (complete response [CR] plus partial response [PR]); secondary efficacy measures included CR, PR, progression-free survival (PFS), and overall survival (OS). Adverse events (AEs) were assessed. ResultsA total 217 patients (≥61.1% male in each group) were included in the analysis (Table). At diagnosis, all patients had an ECOG score of 0–2; most had RAI stage 0-II (16 had stage III/IV) and Binet stage A or B (29 had stage C). Mean number of treatment cycles (28 days/cycle) per group ranged from 5.1 to 5.9. Mean dose per cycle ranged from 133.6 to 165.9 mg/m2 for bendamustine and 391.1 to 412.1 mg/m2 for rituximab in those groups (Table). Median follow-up was 3 years (range 1–5).Observed ORRs were >83% in all groups (Table); 1 patient each in the 60–70 B and 60–70 BR groups had progressive disease, and 1 in the ≥70 BR group was not assessable. By Kaplan-Meier analysis, median PFS and OS have been reached in the 60–70 B (PFS: 14.8 months, OS: 41.0 months) and ≥70 B (PFS: 32.5 months, OS: 40.1 months) groups only. PFS results are shown in the Figure.There were 26 deaths at the time of analysis. The most common grade 3/4 hematologic AEs were febrile neutropenia (n=28) in the ≥70 BR group, leukopenia (n=15) in the ≤60 BR group, and leukopenia (n=25) in the ≥70 BR group. Depression was the most common grade 3/4 nonhematologic AE, affecting all patients in the ≤60 BR and 60–70 B groups, 48/50 patients in the 60–70 BR group, 35/36 in the ≥70 B, and 94/95 in the ≥70 BR. Other common grade 3/4 nonhematologic AEs included fatigue (2 patients in ≥70 BR and 1 patient each in 60–70 B and ≥70 B) and infections/infestations (2 patients in ≤60 BR and 3 in ≥70 BR). Thirty patients were hospitalized (Table). Dose reductions were most frequent in the ≥70 B group (67%) and least in the 60–70 B and 60–70 BR groups (17% and 12.0%, respectively) (Table). Dose delays occurred for 1 patient each in the ≤60 BR, 60–70 B, and ≥70 BR groups and 4 patients in the ≥70 B group. ConclusionsData from this real-world chart review indicate that bendamustine alone or with rituximab provides high response rates and an acceptable safety profile with low rates of dose delay in all patient age groups (≤60, 60–70, and ≥70) with previously untreated CLL. These findings are similar to those reported in large clinical trials.Table<60 BR n=2460–70 B n=1260–70 BR n=50>70 B n=36>70 BR n=95Mean (SD) ageAt diagnosis50.3 (6.4)62.6 (4.9)63.2 (4.3)74.5 (5.6)72.5 (5.5)Start of therapy53.1 (6.2)65.9 (2.1)65.7 (2.5)76.9 (4.8)75.5 (4.5)Mean dose (mg/m2) per cycleBendamustine154.0 (41.5)153.7 (32.5)165.9 (27.0)133.6 (39.0)147.7 (37.6)Rituximab412.1 (107.6)NA392.1 (100.4)NA402.5 (71.4)ResponseORR %10083889790CR5833441937PR4250447853Hospitalizations, patients (%)3 (13)2 (17)6 (12)5 (14)14 (15)Dose reductions, patients (%)9 (38)2 (17)9 (18)24 (67)30 (32)Dose delays, patients (%)1 (4)1 (8)04 (11)1 (1) [Display omitted] Support: Teva Pharmaceutical Industries Ltd. Disclosures:Günther:Mundipharma: Consultancy, Honoraria. Bartels:rgb Onkologisches Management GmbH, which received research funding from Teva Pharmaceuticals: Employment, Research Funding. Sterchele:Teva Pharmaceutical Industries Ltd.: Employment.

The down-regulation of miR-125b in chronic lymphocytic leukemias leads to metabolic adaptation of cells to a transformed state

AbstractMiR-125b-1 maps at 11q24, a chromosomal region close to the epicenter of 11q23 deletions in chronic lymphocytic leukemias (CLLs). Our results establish that both aggressive and indolent CLL patients show reduced expression of miR-125b. Overexpression of miR-125b in CLL-derived cell lines resulted in the repression of many transcripts encoding enzymes implicated in cell metabolism. Metabolomics analyses showed that miR-125b overexpression modulated glucose, glutathione, lipid, and glycerolipid metabolism. Changes on the same metabolic pathways also were observed in CLLs. We furthermore analyzed the expression of some of miR-125b–target transcripts that are potentially involved in the aforementioned metabolic pathways and defined a miR-125b–dependent CLL metabolism-related transcript signature. Thus, miR-125b acts as a master regulator for the adaptation of cell metabolism to a transformed state. MiR-125b and miR-125b–dependent metabolites therefore warrant further investigation as possible novel therapeutic approaches for patients with CLL.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d(+)CD38(+) and CD49d(+)CD38(-) primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d(+)CD38(-) CLL-derived cell line Mec-1. Results indicate that CD49d(+)CD38(+) cells adhered more efficiently onto CD49d-specific substrates than CD49d(+)CD38(-) cells (P < 0.001). Upon adhesion, CD49d(+)CD38(+) cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d(+)CD38(-) cells (P = 0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d(+)CD38(+) cells were more resistant to serum-deprivation-induced (P < 0.001) and spontaneous (P = 0.03) apoptosis than the CD49d(+)CD38(-) counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Abstract 2728 Background.We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods.WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results.The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn’t exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary.A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures:Roccaro:Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Demographics, Treatment Patterns, Safety, and Real-World Effectiveness in Patients ≥70 Years of Age with Chronic Lymphocytic Leukemia Receiving Bendamustine with or without Rituximab,

Abstract 3914 BackgroundBendamustine is a unique, well-established alkylating agent with multifaceted actions leading to cancer cell death in several hematologic malignancies. In a phase 3 trial in treatment-naïve patients with chronic lymphocytic leukemia (CLL), rates of response and progression-free survival (PFS) were significantly superior to those for chlorambucil [Knauf WU et al. J Clin Oncol 2009;27:4378–84]. In vitro studies have found that the cytotoxic activity of bendamustine against CLL-derived cell lines is synergized by rituximab, an anti-CD20 monoclonal antibody [Demidem A et al. Cancer Biother Radiopharm 1997;12:177–86]. Older patients may demonstrate lower tolerance to chemoimmunotherapy [Foon KA & Hallek MJ. Leukemia 2010;24:500–11], and published clinical data on bendamustine-rituximab in CLL are scarce. Thus, this retrospective study sought to characterize a population of adults ≥70 years old with CLL receiving bendamustine with or without rituximab, describe patterns of care, assess data on real-world effectiveness outside of the controlled environment of clinical trials [Waldthaler C et al. Wien Klin Wochenschr 2011;123:269–275], and assess safety. MethodsRecords were extracted from US Oncology iKnowMed (iKM) record databases for all outpatients ≥70 years old with CLL (but no other tumor) and more than 1 visit recorded (but not enrolled in clinical trials) who received bendamustine between March 2008 and May 2010. Patients were classified as treatment-naïve or relapsed (including ≥ second-line therapy). To ascertain mortality, the iKM data were supplemented with vital-status data from the Social Security Administration Death Index. The overall response rate (ORR) included complete response (CR), nodal partial response (nPR), and partial response (PR). PFS was time from first bendamustine dose to progressive disease (change in line of therapy), relapse, or death from any cause. Data from patients who did not die, or had no progression and were lost to follow-up were censored. ResultsAmong 91 patients, the mean (SD) initial age at beginning of first therapy was 77.4 (5.6) years, age at diagnosis was 70.3 (6.5) years, and 63.7% were male. Of the 16 (17.6%) treatment-naïve patients, 10 had received bendamustine monotherapy and 6 received bendamustine-rituximab. Of the 75 (82.4%) relapsed patients, 20 had received bendamustine monotherapy and 55 received bendamustine-rituximab. The observed ORR for treatment-naïve patients was 56.3% (n=9; 18.8% CR, 37.5% PR, and 0 nPR); 6.3% had progressive disease. For relapsed patients, the ORR was 58.7% (n=44; 13.3% CR, 44.0% PR, and 1.3% nPR); 24.0% had progressive disease. Among patients with data, median PFS for 16 treatment-naïve patients has not been reached (median follow-up 15.1 months); for 73 relapsed patients, PFS was 18.4 months. Kaplan-Meier estimates for PFS over time for each group are shown in the Figure. No unexpected toxicities were seen. The overall rate of blood/bone marrow toxicities (all grades) was 40.7%; grade 3/4 rates were 18.8% for treatment-naïve patients and 25.3% for relapsed patients. Other grade 3/4 adverse events (AEs) included upper respiratory infection and abdominal pain in the relapsed group as well as rash and sepsis in both groups (n=1 each). The most frequent nonhematologic AEs (≥5%, any grade) were fatigue (33.0%), weight loss (11.0%), infection (9.9%; herpes zoster [n=2]; cryptococcal sepsis, Klebsiella sepsis, and pneumonia [n=1 each]), gastrointestinal (8.8%), fever (8.8%), pulmonary (6.6%), and rash (5.5%). [Display omitted] o = data censored. ConclusionsIn this retrospective chart review of patients ≥70 years old with CLL, bendamustine, either alone or with rituximab, provided meaningful response rates and was generally well tolerated. The length of PFS of both treatment-naïve and relapsed patients was clinically meaningful.This research was sponsored by and conducted in collaboration with Cephalon, Inc., Frazer, PA. Disclosures:Sterchele:Cephalon, Inc.: Employment. Beygi:Cephalon, Inc.: Employment. Kennealey:Cephalon, Inc.: Employment.