Therapeutic Strategy For Chronic Lymphocytic Leukemia Research Articles

Targeting the MDM2-p53 Interaction with Siremadlin: A Promising Therapeutic Strategy for Treating TP53 Wild-Type Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) treatment has transitioned from traditional chemotherapy to more targeted therapies, but challenges such as resistance and suboptimal responses persist. This study aimed to evaluate HDM201, a second-generation MDM2-p53 binding antagonist, as a novel therapeutic strategy for CLL, with a focus on its effectiveness across different TP53 genetic contexts. Methods: We utilized a panel of B cell leukemia-derived cell lines with varying TP53 statuses, including TP53-knockout (KO) derivatives of the human B cell line Nalm-6, and assessed the impact of HDM201 on primary CLL samples with both TP53 wild-type and mutant backgrounds. Results: Our results revealed that TP53 wild-type and heterozygous TP53-KO Nalm-6 cells were sensitive to HDM201, whereas homozygous TP53-KO cells and B cells with TP53 mutations exhibited significant resistance. Resistance was also noted in primary CLL samples with TP53 mutations. HDM201 effectively stabilized p53 and induced apoptosis in TP53 wild-type cells but had limited efficacy in TP53 mutant cells. Conclusions: These findings indicate that HDM201 holds promise as an additional targeted therapy option for wild-type TP53 CLL. The results underline the importance of TP53 status in predicting treatment efficacy and highlight the potential of HDM201 as a valuable addition to explore in CLL therapy. Future research should focus on identifying additional biomarkers of response and exploring the optimal way to include HDM201 in combination therapies to improve treatment outcomes in CLL.

A CX3CR1 (fractalkine receptor) Small Molecular Antagonist (KAND567) Suppressed the Growth Promoting Effect of Monocytes on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia worldwide. Novel treatment strategies are needed to improve the prognosis. Non-malignant cells of the tumor microenvironment (TME) are of importance for the growth of tumor cells. Monocytes expressing various chemokine receptors as CX3CR1 (the fractalkine receptor (FKNR)) have been shown to promote the growth of tumor cells in vitro and in vivo. In CLL, monocyte derived nurse-like cells (NLC) support the growth of CLL cells, promoting leukemic cell proliferation and survival by secretion of chemokines/cytokines. In this study, we studied the role of autologous monocytes on the survival of CLL cells and the effects of a small molecule antagonist of CX3CR1 (KAND567/FKNRa). KAND567 is in clinical trials for diseases with an activated fractalkine system as part of the pathobiology including covid-19 infected patients as well as patients with acute myocardial infarction and ovarian carcinoma. Methods: Primary CLL cells and B cells (CD19 +) of healthy donors as well as autologous monocytes (CD14 +) from CLL patients (n=22) and healthy control donors (n=11) were cultured alone or in co-cultures (isolated B cells together with autologous monocytes) with the FKNRa (250 and 1000 nM) for up to 120 h. Apoptosis of CD19 + cells and monocytes was analysed by Annexin V/PI staining, Western blotting and the IncuCyte® Live Cell Imaging System. The NBT assay was also applied to check morphological changes of monocytes. Plasma concentration of fractalkine (CX3CL1) was determined by ELISA. Results and discussion: CLL patients had a significant (p=0.0001) increase in the plasma concentration of CX3CL1 compared to healthy controls. CLL cells as well as normal B cells did not express surface CX3CR1. However, monocytes from both CLL patients and healthy donors expressed high levels of surface CX3CR1. A significantly higher proportion of pro-inflammatory intermediate (CD14 +/CD16 +) and non-classical (CD14 -/CD16 +) monocytes (p<0.0001 and <0.01 resp.) as well as intermediate and non-classical CX3CR1 + monocytes were observed in patients with active CLL compared to healthy donors (p<0.0001 and <0.01 resp.). Moreover, CX3CR1 + intermediate and non-classical monocytes were significantly higher in CLL patients with active disease compared to those with early-stage disease (p<0.0001 and <0.001 resp.). A growth supportive effect of autologous monocytes was observed on survival of both CLL cells and healthy donor B cells in 120 h cell co-culture experiments (p<0.001 and <0.01 resp.). FKNRa reduced the number of alive CLL cells in a dose-dependent manner in co-cultures with autologous monocytes (p<0.05). No effect was seen on CD19 + B cells of healthy donors co-cultured with autologous monocytes. FKNRa did not induce apoptosis of CLL cells alone. Furthermore, FKNRa also inhibited transformation of CLL monocytes to NLC (p<0.05) which was not the case for normal monocytes. Conclusion: The CX3CR1/CX3CL1 (fractalkine receptor/ligand)) axis appears to be activated in CLLand might be of importance for the NLC-driven growth of CLL cells. This is the first report of a small molecule antagonist of CX3CR1 reducing the growth supportive effect of autologous CLL monocytes on leukemic CLL cells. Disruption of growth promotion induced by monocytes may prevent leukemic cell proliferation and survival. Inhibition of autologous monocytes in the TME might represent a new therapeutic strategy in CLL, complementary to treatment with drugs that directly target the tumor cells.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL

AbstractDysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5′ untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation–related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

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

Activation of Interferon Signaling in Chronic Lymphocytic Leukemia Cells Contributes to Apoptosis Resistance via a JAK-Src/STAT3/Mcl-1 Signaling Pathway

Besides their antiviral and immunomodulatory functions, type I (α/β) and II (γ) interferons (IFNs) exhibit either beneficial or detrimental effects on tumor progression. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal CD5+ B lymphocytes that escape death. Drug resistance and disease relapse still occur in CLL. The triggering of IFN receptors is believed to be involved in the survival of CLL cells, but the underlying molecular mechanisms are not yet characterized. We show here that both type I and II IFNs promote the survival of primary CLL cells by counteracting the mitochondrial (intrinsic) apoptosis pathway. The survival process was associated with the upregulation of signal transducer and activator of transcription-3 (STAT3) and its target anti-apoptotic Mcl-1. Furthermore, the blockade of the STAT3/Mcl-1 pathway by pharmacological inhibitors against STAT3, TYK2 (for type I IFN) or JAK2 (for type II IFN) markedly reduced IFN-mediated CLL cell survival. Similarly, the selective Src family kinase inhibitor PP2 notably blocked IFN-mediated CLL cell survival by downregulating the protein levels of STAT3 and Mcl-1. Our work reveals a novel mechanism of resistance to apoptosis promoted by IFNs in CLL cells, whereby JAKs (TYK2, JAK2) and Src kinases activate in concert a STAT3/Mcl-1 signaling pathway. In view of current clinical developments of potent STAT3 and Mcl-1 inhibitors, a combination of conventional treatments with these inhibitors might thus constitute a new therapeutic strategy in CLL.

BTK inhibitors and anti-CD20 monoclonal antibodies for treatment-naïve elderly patients with CLL.

Older patients account for the majority of patients with chronic lymphocytic leukemia (CLL), and so strategies for managing CLL in this population is of upmost importance. Inhibition of Bruton’s tyrosine kinase (BTK) has been a successful therapeutic strategy in CLL, and the first-generation BTK inhibitor ibrutinib has been shown to be superior to standard chemoimmunotherapy in multiple studies specifically targeting older patients. A second-generation BTK inhibitor, acalabrutinib, has also been studied in CLL, and has recently been granted breakthrough designation by the United States Food and Drug Administration. One ongoing question is whether the addition of anti-CD20 monoclonal antibodies improve response or response durability with BTK inhibitors. In this review, we will discuss clinical trials of ibrutinib and acalabrutinib in older patients with CLL, and the possible contributions of anti-CD20 antibodies to these therapies.

Preliminary Results of Ibrutinib Followed By Ofatumumab Consolidation in Previously Untreated Patients with Chronic Lymphocytic Leukemia (CLL): GELLC7 Trials from the Spanish Group of CLL (GELLC)

Preliminary Results of Ibrutinib Followed By Ofatumumab Consolidation in Previously Untreated Patients with Chronic Lymphocytic Leukemia (CLL): GELLC7 Trials from the Spanish Group of CLL (GELLC)

Highly Potent BTK Degradation Induced By NRX0492 As a Therapeutic Strategy for CLL

B-cell receptor (BCR) signaling plays an essential role in normal B-cell development and is a key driver of proliferation and survival of chronic lymphocytic leukemia (CLL) B cells. BCR signals through BTK to NF-ĸB. Targeted inhibition of BTK with ibrutinib, a first-in-class covalent BTK inhibitor, is highly effective in treating CLL and related B-cell malignancies. However, progressive disease on continuous therapy with ibrutinib has been associated with mutations in BTK and PLCG2. Most patients with acquired resistance and progressive CLL on ibrutinib have mutations in BTK affecting the cysteine at position 481 (C481) to which ibrutinib covalently binds. The common C481S mutation increases IC50 with ibrutinib from low nM to μM concentrations. The emergence of specific BTK mutations in ibrutinib resistant CLL further validate BTK as a valuable therapeutic target in CLL. Therefore, agents that can target C481 mutant BTK are of great interest, and non-covalent BTK inhibitors have entered clinical trials. Here we investigated an alternative approach using a chimeric targeting molecule: NRX0492. NRX0492 combines two molecules: a "hook" that binds BTK with a "harness" that recruits ubiquitin ligases that mediate proteasome-dependent degradation of BTK. In cell line models, NRX0492 induce both wild-type and C481S mutant BTK degradation efficiently and specifically. Here we sought to define the on-target effects of NRX0492 in primary CLL cells in vitro and in vivo and to test NRX0492 against ibrutinib-resistant primary CLL cells. CLL cells were treated at densities of 5 × 106 cells/ml in 24-well plates with NRX0492, ibrutinib, the "hook" moiety devoid of the ubiquitin ligase binding moiety, or DMSO. BTK levels were quantified by Western blotting and flow cytometry. The mean ED50 and ED90 of NRX0492 for BTK degradation at 4 hours was 0.18nM and 0.5nM, respectively. At ED90 concentrations, mean cell viability of cultures treated with NRX0492 was 97.5%, and not different from viability of controls. Incubation at ED50 up to 24 hours also did not induce cell death. A lack of substantial cytotoxic activity with BTK degradation is in line with observations with BTK inhibitors under similar conditions. BTK degradation was achieved rapidly; in time-course experiments of CLL cells treated with 0.2nM NRX0492 the mean half-live of BTK protein was 2.7 hours (range 2.4h-3h), and at 2nM 99% of BTK was degraded within 4 hours. Next, we tested the rate of recovery of BTK expression after drug washout. After 0.2nm NRX0492 treatment for 4 hours, mean BTK levels were 55% of pre-treatment. After washout and continued culture of cells in complete medium for 96 hours, BTK levels were 41% of pre-treatment. In additional experiments with washout of NRX0492 after 4 hours, we found the lowest levels of BTK 48hours post-treatment, indicating a sustained drug effect. There was no significant difference in the efficacy of BTK degradation between CLL samples subdivided by IGHV mutation status or FISH categories. At 0.2nM NRX0492 for 4 hours, mean remaining BTK was 54% in IGHV mutated and 55% in IGHV unmutated samples (P =0.78), and 60% vs 50% for del(13q) vs del(17p) (P =0.18). Next, we studied samples from two patients progressing on ibrutinib with classic C481S mutations at cancer cell fractions of 50% and 84%, respectively. Both patients remained on ibrutinib at the time of sample collection. After 4 hours of treatment with 0.2nM NRX0492, BTK levels were 50% and 15% of DMSO-treated controls, respectively, consistent with degradation of mutant but not ibrutinib bound BTK. Wild-type BTK bound by ibrutinib is not degraded because ibrutinib prevents NRX0492 binding to BTK. In conclusion, we find NRX0492 is highly effective in degrading BTK in CLL cells. NRX0492 is effective at sub nM concentrations with ED50 ~0.2nM and ED90 ~0.5nM in primary CLL cells. Onset of BTK degradation was rapid and sustained for days after drug washout. NRX0492 was effective across CLL risk groups and equally effective in degrading wild-type BTK and C481S-mutant BTK. Studies testing NRX0492 in vivo in PDX models are ongoing and will inform dosing regimens for clinical studies. Disclosures Kelly: Nurix Therapeutics: Employment. Robbins:Nurix Therapeutics: Employment. Tan:Nurix Therapeutics: Employment. Ingallinera:Nurix Therapeutics: Employment. Sands:Nurix Therapeutics: Employment. Baskar:NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Wiestner:Pharmayclics: Research Funding; Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding.

Three Different Jak2/Stat3-Related Pathways Favor the Survival of Chronic Lymphocytic Leukemia Neoplastic Clone

INTRODUCTIONChronic lymphoproliferative disorders are characterized by the expansion of malignant lymphocytes, the most common form being Chronic Lymphocytic Leukemia (CLL). Besides intrinsic abnormalities, the acquisition of the transformed phenotype and the diffusion of disease are related to the favorable cross-talking tumor cell-microenvironment. Furthermore, it has been demonstrated that CLL cells own an amplified mitochondrial respiration leading to an increased Reactive Oxygen Species (ROS) production and intrinsic oxidative stress. Several molecules released by microenvironmental partners signal through JAK-STAT pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. We previously analyzed STAT3 and JAK2 expression, phosphorylation and localization in normal and leukemic B cells, demonstrating an abnormal activation of this pathway in the neoplastic clone with respect to normal B lymphocytes. We focused on STAT3 constitutive phosphorylation at serine (Ser) 727 residue since it has been recently observed that the presence of mt-STAT3 Ser727 promotes mitochondrial respiration and, generally, cell viability. We hypothesized the involvement of JAK2/STAT3 axis in 3 different pathways: (i) canonical “IL-6 related pathway”; (ii) JAK2/STAT3 - BCR/Lyn crosstalk; (iii) STAT3 effects on mitochondrial regulation.METHODSSTAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC). Purified cells (2x106 cells/ml) were cultured, and treated with the JAK2 inhibitor AG490 (10, 50 and 100μM) and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs and with/without Ibrutinib (2.5μM) and Venetoclax (1nM). CLL and normal B cell viability was tested with Annexin V/PI test by FC. P-STAT3 Ser727 expression has been correlated with p44/42, SAPK-JNK, NF-kB p65 and p38 MAPK activation by Reverse Phase Protein Microarrays (RPPA) in 57 therapy-free patients presenting good (mutated IGHV, absence of CD38 or normal karyotype) and poor (unmutated IGHV, CD38 expression or 17p deletion) prognostic factors.RESULTSWe found that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. We demonstrated that STAT3 and JAK2 were similarly overexpressed in both good and poor prognosis CLL patients. However, a significant correlation between STAT3 expression levels and their overall survival was observed. Considering STAT3 over-expression and its correlation with the clinical outcome in CLL and, according to our hypothesis, we moved forward with the analysis of the three different JAK2/STAT3 pathways. We demonstrated that AG490 and Stattic were able to induce a dose-dependent apoptosis in CLL cells, also bypassing environmental protection. AG490, targeting JAK2, inhibited the phosphorylation of SHP-1 at Ser591, activating the phosphatase. In turn, SHP-1 activation led to Lyn Tyr396 dephosphorylation/inactivation. Treatment with Stattic did not affect Lyn and SHP-1 phosphorylation since this inhibitor acts downstream to AG490. In fact, simultaneous administration of Ibrutinib led to an increase of apoptosis only in Stattic, but not in AG490 treated cells. This confirms a possible dual role of JAK2 inhibition. Venetoclax/AG490 and Venetoclax/Stattic co-treatment did not show an increased cell death rate, consistent with a downstream effect of Venetoclax to both AG490 and Stattic. RPPA analysis demonstrated a correlation between STAT3, Ser727 constitutively phosphorylated in CLL, and P-p44/42 Thr202/Tyr204, P-SAPK-JNK Thr-183/Tyr185, NF-kB p65 Ser-536 and p38 MAPK Thr-180/Tyr-182 expression. All these proteins are described as involved in an alternative pathway that allows STAT3 Ser727 to exert a pro-survival effect thorough the regulation of mitochondrial activity.CONCLUSIONSThe analysis of JAK2 and STAT3 expression, activation and inhibition lets us to highlight the importance of JAK2/STAT3 axis in the maintenance of 3 different survival pathways in CLL B cells. Furthermore, the correlation we demonstrated with clinical outcome of patients and the strengthening of Ibrutinib effect when we targeted this axis could represent a starting point for the development of new therapeutic strategies in CLL. DisclosuresTrentin:Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees.

Lysosomotropic agents selectively target chronic lymphocytic leukemia cells due to altered sphingolipid metabolism.

Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL.

Activity of PIM Kinases in Chronic Lymphocytic Leukemia Modulates Tumor Cell Survival and Stromal Interactions through a Pleiotropic Mechanism Involving Modulation of CXCR4 - mTOR Pathway

The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells.In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p<0.001). PIM2 expression was higher in patients with advanced disease (Rai 3-4, p=0.004). Higher PIM2 expression was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood (p=0.024 and p=0.0059, respectively; Herishanu et al., 2010), suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of PIM kinases in these processes, we assessed whether PIM inhibition interferes with CXCR4 - mediated signaling and migration. We incubated primary CLL cells with a novel pan-PIM inhibitor SEL24-B489 and found decreased phospho-CXCR4 (Ser339) level and decreased CXCR4 surface expression. Primary CLL cells incubated with with SDF1 (500 ng/ml, 15-60 min) exhibited highly increased phosphorylations of mTOR (Ser2448) and Akt (Ser437). Since PIM kinases modulate mTOR signaling, we further investigated whether inhibition of PIM kinases with SEL24-B489 interferes with CXCR4/mTOR pathway. SEL24-B489 blocked baseline phosphorylation level of mTOR negative regulator p-TSC2 (Ser1798). Since TSC2 Ser1798 phosphorylation relieves its suppression on RHEB and promotes mTOR activity, we next assessed PIM inhibitor - induced changes in p-mTOR (Ser2448). Upon SEL24-B489 treatment, mTOR Ser2448 phosphorylation and activity of mTOR downstream substrates (p-Akt Ser473 and p-4EBP1 Thr37/46) markedly decreased. Pre-incubation of CLL cells with 10 uM SEL24-B489 or 10 uM mTOR inhibitor OSI-027 before SDF1 treatment restrained the increase of p-mTOR (Ser2448), p-Akt (Ser473), p-4EBP1 (Thr37/46) and p-TSC2 (Ser1798), and consequently impaired CLL cells migration in the SDF1 gradient. In 4 out of 7 analyzed patients the effect of SEL24-B489 on CLL migration was stronger than the effect of OSI-027 and in the remaining 3 patients, the effects of both inhibitors were similar. Since interactions of CLL cells with their microenvironment block the cytotoxicity of chemotherapeutic agents, we next compared the apoptotic response to SEL24-B489 in CLL cells cultured in the absence or presence of human bone marrow HS5 cells. CLL cells were seeded on HS5 monolayers and treated with SEL24-B489 (5 uM and 10 uM) for 48 hours. In 6 out of 7 cases SEL24-B489 overcame the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.Taken together, our data demonstrate that CXCR4/SDF1 signal in chronic lymphocytic leukemia cells is transduced through mTOR pathway and that CXCR4 - triggered mTOR activity is modulated by PIM kinases. Pan-PIM inhibitor SEL24-B489 decreased CXCR4 surface expression and SDF-1 - triggered mTOR activity. Finally, SEL24-B489 decreased protective effects of tumor microenvironment and induced CLL cells apoptosis even in the presence of stromal cells. Since overexpression of PIM kinases might be associated with adverse clinical characteristics at diagnosis, PIM inhibition might be a rational therapeutic strategy in CLL. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

The MEK1/2 inhibitor, MEKi-1, induces cell death in chronic lymphocytic leukemia cells under conditions that mimic the tumor microenvironment and is synergistic with fludarabine

The Raf-1/MEK/ERK1/2 pathway has become a focus for novel cancer therapies. This study sought to investigate whether targeting MEK1/2 may represent a therapeutic option for chronic lymphocytic leukemia (CLL). The MEK1/2 inhibitor, MEKi-1, induced apoptosis of CLL cells and was synergistic with fludarabine under conditions that mimic the tumor microenvironment, irrespective of poor-risk characteristics. MEKi-1 down-regulated the activities of AKT and ERK1/2 and was synergistic with fludarabine through a mechanism that involved potentiation of DNA damage and attenuation of the activity of ERK1/2 and expression of Mcl-1. This study highlights the significant role of the mitogen-activated protein kinase (MAPK)–ERK1/2 pathway in mediating the effects of the CLL tumor microenvironment and suggests that targeting MEK1/2 in CLL cells may impact upon the activity of both ERK1/2 and AKT. Inhibitors of MEK1/2 as single agents or in combination with DNA-damaging agents may represent a novel therapeutic strategy for CLL.

Dual Inhibition of PIM and PI3-Kinase By Ibl-202 Is Highly Synergistic Compared to Mono-Molecular Inhibition and Represents a Novel Treatment Strategy for Chronic Lymphocytic Leukemia

BACKGROUND Trials of ibrutinib and idelalisib for Chronic Lymphocytic Leukemia (CLL) targeting Btk and PI3-kinase respectively, illustrate the potential of targeting components of the B-cell receptor (BCR) signalling pathway. Emerging evidence suggests that subgroups of CLL patients develop resistance to these agents. In particular late relapses on ibrutinib appear to be associated with a high frequency of acquired mutations in Btk and PLCg2. Identification of novel combination therapies or agents that target multiple molecules in key signaling pathways represents a rational approach in the development of novel treatment strategies. Pim (provirus integration site for Moloney murine leukemia virus) family proteins are proto-oncogenic and involved in B-cell development and lymphoid malignancies. They are highly conserved serine/threonine kinases and are overexpressed in CLL. Given the clinical efficacy of idelalisib and results of preclinical studies of the PIM kinase SGI-1776 [Chen et al., 2009], we sought to investigate the potential of simultaneous inhibition of PIM and PI3-kinase for CLL therapy.METHODS The effects of the novel inhibitor of PIM kinase, pPIMi, and the dual inhibitor of PIM/PI3-kinase, IBL-202 (both from Inflection Bioscience Ltd, Ireland), were investigated against panels of primary CLL patient samples and haematological lines. The in vitro efficacy of both agents was compared to that of SGI-1776 and idelalisib and studied under conditions involving CLL cell co-culture using CD40L-expressing fibroblasts that mimic the support offered by the CLL tumor microenvironment.RESULTS Inhibition of PIM kinase by pPIMi was as effective as SGI-1776, inducing a dose dependent decrease in CLL-cell viability. The rationale for dual inhibition of PIM and PI3-kinase was investigated by combining pPIMi with idelalisib and by calculation of combination indices (CI), where CI values of < 1 are indicative of synergy. In primary CLL patient samples (Figure 1) and 3 B-cell lines (Mec1, Ramos and Raji) the combination proved highly synergistic, with CI values of 0.07 + /- 0.03 and 0.11 +/- 0.07 respectively, for fractional cell killing effects of between 0.1 and 0.9.Consistent with our assessment of synergy between pPIMi and idelalisib, dual inhibition of PIM and PI3-kinase by IBL-202 was significantly more effective at inducing cell death than either pPIMi or idelalisib as single agents (Figure 2), evidenced by the significantly lower IC50 against CLL patient samples (7.47 µM, 43.12 µM, 1.23 µM for pPIMi, idelalisib and IBL-202 respectively).The rationale for targeting PIM and PI3-kinase using IBL-202 was further highlighted by culturing CLL cells in the presence of a CD40L-expressing fibroblast monolayer, which represents a model of the tumor microenvironment. CD40L fibroblast co-culture significantly increased CLL-cell survival under control conditions but also conferred resistance to the PIM inhibitor pPIMi. In contrast CLL cells cultured under these conditions were sensitive to IBL-202 in a dose dependent manner.CONCLUSION Inhibition of PIM and PI3-kinase is potently synergistic against CLL cells and is particularly effective under conditions that mimic the tumor microenvironment. Dual targeting of PIM and PI3-kinase by IBL-202 may represent a novel therapeutic strategy for CLL that minimizes the risk of acquired mutations associated with mono-molecular inhibition of BCR-signaling components. [Display omitted] [Display omitted] DisclosuresO’Neill:Inflection Biosciences: Employment, Equity Ownership. O’Dwyer:Inflection Biosciences Ltd: Membership on an entity’s Board of Directors or advisory committees. Mulligan:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

The gene expression response of chronic lymphocytic leukemia cells to IL-4 is specific, depends on ZAP-70 status and is differentially affected by an NFκB inhibitor.

Interleukin 4 (IL-4), an essential mediator of B cell development, plays a role in survival of chronic lymphocytic leukemia (CLL) cells. To obtain new insights into the function of the IL-4 pathway in CLL, we analyzed the gene expression response to IL-4 in CLL and in normal B cells (NBC) by oligonucleotide microarrays, resulting in the identification of 232 non-redundant entities in CLL and 146 in NBC (95 common, 283 altogether), of which 189 were well-defined genes in CLL and 123 in NBC (83 common, 229 altogether) (p<0.05, 2-fold cut-off). To the best of our knowledge, most of them were novel IL-4 targets for CLL (98%), B cells of any source (83%), or any cell type (70%). Responses were significantly higher for 54 and 11 genes in CLL and NBC compared to each other, respectively. In CLL, ZAP-70 status had an impact on IL-4 response, since different sets of IL-4 targets correlated positively or negatively with baseline expression of ZAP-70. In addition, the NFκB inhibitor 6-Amino-4-(4-phenoxyphenethylamino)quinazoline, which reversed the anti-apoptotic effect of IL-4, preferentially blocked the response of genes positively correlated with ZAP-70 (e.g. CCR2, SUSD2), but enhanced the response of genes negatively correlated with ZAP-70 (e.g. AUH, BCL6, LY75, NFIL3). Dissection of the gene expression response to IL-4 in CLL and NBC contributes to the understanding of the anti-apoptotic response. Initial evidence of a connection between ZAP-70 and NFκB supports further exploration of targeting NFκB in the context of the assessment of inhibition of the IL-4 pathway as a therapeutic strategy in CLL, especially in patients expressing bad prognostic markers.

MicroRNAome Expression in Chronic Lymphocytic Leukemia: Comparison with Normal B-cell Subsets and Correlations with Prognostic and Clinical Parameters

Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial. We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood. Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone-like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival. This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL.

Poly(ADP-ribose) polymerase inhibitor CEP-8983 synergizes with bendamustine in chronic lymphocytic leukemia cells in vitro

DNA repair aberrations and associated chromosomal instability is a feature of chronic lymphocytic leukemia (CLL). To evaluate if DNA repair insufficiencies are related to methylation changes, we examined the methylation of nine promoter regions of DNA repair proteins by bisulfide sequencing in 26 CLL primary samples and performed quantitative PCR on a subset of samples to examine BRCA1 expression. We also investigated if changes in cytogenetic or expression level of DNA repair proteins led to changes in sensitivity to a novel PARP inhibitor, CEP-8983, alone and in combination with bendamustine. No changes in promoter methylation were identified in BRCA1, BRCA2, FANC-C, FANC-F, FANC-L, ATM, MGMT, hMLH1 and H2AX except for two cases of minor BRCA1 hypermethylation. CLL samples appeared to have reduced BRCA1 mRNA expression uniformly in comparison to non-malignant lymphocytes irrespective of promoter hypermethylation. CEP-8983 displayed single agent cytotoxicity and the combination with bendamustine demonstrated synergistic cytotoxicity in the majority of CLL samples. These results were consistent across cytogenetic subgroups, including 17p deleted and previously treated patients. Our results provide rationale for further exploration of the combination of a PARP inhibitor and DNA damaging agents as a novel therapeutic strategy in CLL.

In Vitro and in Vivo Targeting of Chronic Lymphocytic Leukemia Using CX-4945, a Clinical-Stage CK2-Specific Inhibitor.

AbstractAbstract 2897Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma.We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P<0.0001 for each dose, 2-tailed paired Student’s t test), as determined by Annexin V-APC/7-AAD staining. Moreover, the effect of CX-4945 was time- and dose-dependent in 4 out of 4 cases that were more thoroughly analyzed. Similar results were obtained using MEC1, MEC2, WaC3CD5, JVM3 and MO1043 cell lines whose IC50 ranged between 3.1 and 5.8μM. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival, it did not prevent CX-4945-mediated apoptosis of CLL cells. Most importantly, CX-4945 induced a stronger decrease in the viability of CLL cells from patients with higher percentage of malignant cells in the blood (R2=0.4176, P=0.0317, n=11, Pearson correlation), Binet stage B/C (P=0.0424, n=10, 2-tailed unpaired Student’s t test) or higher plasma β2 microglobulin levels (P=0.0239, n=9). Furthermore, CLL cells with a higher proliferation rate (LDT < 12 months) were also more sensitive to CX-4945 (P=0.0007, n=11). In accordance, the need for treatment positively correlated with the sensitivity to CX-4945 (R2=0.4504, P = 0.0238). These observations suggest that treatment with CK2 inhibitors may be especially beneficial to patients with more advanced or aggressive disease.The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P<0.0001, 2-way ANOVA). Notably, CX-4945 was as effective as fludarabine when used as a single agent, and the combination of the two drugs was significantly more effective than fludarabine alone (P=0.0375). All treatments were well tolerated as evidenced by the maintenance of body weight and the inexistence of signs of overt toxicity.Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL. Disclosures:Stansfield:Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.

γ‐Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and notch down‐regulation

γ-Secretase inhibitors (GSIs) have been proposed for combined therapies of malignancies with a dysregulated Notch signaling. GSI I (Z-Leu-Leu-Nle-CHO) induces apoptosis of some tumor cells by inhibiting proteasome and Notch activity. Alterations in these two cell survival regulators contribute to apoptosis resistance of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the mechanisms whereby GSI I increases apoptosis of primary CLL cells. Time-course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress apoptotic signaling, and a consistent Noxa protein up-regulation. These events precede, and some of them contribute to, mitochondrial alterations, which occur notwithstanding Mcl-1 accumulation induced by GSI I. In CLL cells, GSI I inhibits Notch1 and Notch2 activation only in the late apoptotic phases, suggesting that this event does not initiate CLL cell apoptosis. However, Notch inhibition may contribute to amplify GSI I-induced CLL cell apoptosis, given that Notch activation sustains the survival of these cells, as demonstrated by the evidence that both Notch1 and Notch2 down-regulation by small-interfering RNA accelerates spontaneous CLL cell apoptosis. Overall, our results show that GSI I triggers CLL cell apoptosis by inhibiting proteasome activity and enhancing ER stress, and amplifies it by blocking Notch activation. These findings suggest the potential relevance of simultaneously targeting these three important apoptosis regulators as a novel therapeutic strategy for CLL.

The role of B cell receptor stimulation in CLL pathogenesis.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults in the Western world and is characterized by the accumulation of monoclonal CD5(+) mature B cells in the blood. The disease has a highly variable clinical course. CLL is subdivided into two disease subtypes, whereby leukemias with hypermutated immunoglobulin heavy chain variable (IGHV) genes have a more favorable prognosis than those with unmutated IGHV genes, which tend to show advanced, progressive disease, adverse cytogenetic features and resistance to therapy. The current view is that both CLL types derive from antigen-experienced cells. Based on the finding that the IGHV repertoire is highly restricted and biased, as compared to the normal adult B-cell repertoire, it has been hypothesized that CLL cells are selected by some sort of antigenic pressure. Hereby, either autoantigens or antigens derived from apoptotic cells or pathogens are essential to trigger CLL pathogenesis. Although different cytogenetic aberrations were shown to contribute to CLL leukemogenesis, it remains unclear which abnormalities are primary events. Very recently, whole-genome sequencing identified genes that are recurrently mutated and provided novel insights into the mechanisms of oncogenic transformation. Because of the impact on prognosis, it is important to unravel the role of antigenic selection in CLL. Interestingly, B cell receptor (BCR) signaling is aberrantly increased in CLL and expression of tyrosine kinase ZAP70, which is able to signal downstream of the BCR, is a prognostic indicator. In this context we discuss the functional significance of antigenic selection in CLL and describe emerging agents to target BCR signaling that are currently being tested as a novel therapeutic strategy for CLL.