MAPK-ERK Pathway Activation: The Achilles' Heel of IL-7R‒Induced Steroid Resistance

INTRODUCTION. Mutations in the IL-7R signaling pathway (e.g.IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS or AKT) are associated with steroid resistance and inferior relapse-free survival. These mutant molecules drive strong activation of the JAK-STAT, MAPK-ERK and/or PI3K-AKT signaling pathways. Also, activation of wild type RAS or AKT-reflecting physiological IL7-induced signaling-drives steroid resistance. These observations extend the findings in preB-ALL where RAS mutations were related to steroid resistance. Impaired transcription of pro-apoptotic BIM-an essential direct transcriptional target gene of the glucocorticoid receptor NR3C1 that mediates steroid-induced apoptosis-is observed in various steroid resistant PDX models. AIM. Given the central role of BIM in steroid-induced apoptosis, we investigated how mutations in IL7R or downstream signaling components would affect the function of BIM and other BCL2 protein family members (BCL2, BCLXL and MCL1) in steroid-exposed T-ALL. RESULTS. We generated doxycycline-inducible SUP-T1 and P12 Ichikawa cell line models that can overexpress specific IL-7R signaling mutants. Expression of mutant JAK1, NRAS or IL7Ra molecules results in robust activation of the MAPK-ERK pathway that confers steroid resistance. As a target of NR3C1, BIM transcription was not impaired in these steroid treated cell lines. Therefore, we investigated post-transcriptional mechanisms of steroid resistance. We observed strong phosphorylation of BIM downstream of MAPK-ERK activation under steroid treated and untreated conditions. Exposure to each of three MEK-inhibitors CI1040, Selumetinib or Trametinib abolished not only phospho-ERK but also phosphorylation of BIM. To study the consequences of BIM phosphorylation in relation to binding and inactivation of anti-apoptotic proteins, we immunoprecipitated BIM. Increased BIM levels following steroid exposure was related to increased binding to BCL2, BCLXL and MCL1 in steroid-sensitive control cells. In contrast, activation of mutant JAK1 and NRAS molecules resulted in phosphorylated BIM and reduced binding of BIM to these anti-apoptotic BCL2 family members. This indicates that preventing ERK-mediated phosphorylation of BIM enhances steroid responsiveness. T-ALL patients with activating IL-7R signaling mutations may therefore benefit from treatment combining steroids and MEK inhibitors. Indeed we observed strong synergy between MEK inhibitors and steroids in primary T-ALL patient samples. CONCLUSIONS AND FUTURE PERSPECTIVES. Treatment with MEK-inhibitors Selumetinib or Trametinib was shown effective in reversing steroid resistance, and therefore strongly synergize with steroid treatment. Our data highlights the importance of the dynamic interplay between BIM and anti-apoptotic BCL2 family members in MAPK-ERK-driven steroid resistance. Therefore, we are exploring whether MEK inhibitors (that prevent phosphorylation and inactivation of BIM) combined with BH3-mimetics (that block anti-apoptotic family members) will further enhance restoration of steroid responsiveness in IL-7R signaling mutant T-ALL patients. As physiological IL7-induced signaling is sufficient to raise cellular steroid resistance, these inhibitors could be applied for all IL-7R signaling-dependent T-ALL patients, also in the absence of IL-7R signaling mutations. Refractory/relapse T-ALL patients are now eligible for the current phase 1/2 MEK-inhibitor‒dexamethasone (SeluDex) trial. We are using mass-spectrometry to determine the exact phospho-sites in BIM that drives steroid resistance that can serve as a valuable biomarker. Disclosures No relevant conflicts of interest to declare.

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Differential Expression and Functions of NOTCH Family Members and Involvement of NR4A1 in the Regulation of Apoptosis in CLL

Blood ◽

10.1182/blood.v120.21.3919.3919 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3919-3919

Author(s):

Rainer Hubmann ◽

Martin Hilgarth ◽

Susanne Schnabl ◽

Elena Ponath ◽

Dita Demirtas ◽

...

Keyword(s):

Cell Viability ◽

Mrna Expression ◽

Target Gene ◽

Conflicts Of Interest ◽

Family Members ◽

Dependent Manner ◽

Rt Pcr ◽

Activated State ◽

Induced Apoptosis ◽

The Individual

Abstract Abstract 3919 Chronic lymphocytic leukemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC) dependent manner linking NOTCH2 to the activated state of the leukemic cells. The transcriptional activity of NOTCH2 is associated with the expression of CD23 and enhanced CLL cell viability. However, the regulation and possible functions of the individual NOTCH family members (NOTCH1–4) in CLL cells remain to be clarified. We took advantage of targeting nuclear NOTCH2 using the recently identified NOTCH2 transactivation inhibitor gliotoxin (WO 2006/135949). We also analysed the regulation and possible function of NOTCH1–4 in PKC stimulated CLL cells using a PMA model (Hubmann et al., BJH 2010) and a microenvironment model where CLL lymphocytes were co-cultured with primary bone marrow stromal cells (BMSC) (Shehata et al., BLOOD 2010). Electrophoretic mobility shift assays (EMSA) demonstrated that gliotoxin inhibited DNA-bound NOTCH2 complexes in PMA stimulated CLL cells in parallel to increasing the rate of apoptosis (mean±SD: 67±31% in gliotoxin treated cells versus 13±14% in the untreated controls, n=21). This was associated with downregulation of CD23A mRNA expression and CD23 surface expression (mean±SD: 42±32% versus 83±17%, n=21) as assessed by RT-PCR and FACS analysis. Exceptionally, one CLL case with a recently described NOTCH1 gain of function mutation appeared to be less sensitive to gliotoxin and had a persistent high expression of CD23. We next tested whether NOTCH2 inhibition by gliotoxin is a selective process or indirectly mediated by effects on proteasome regulated apoptosis. Proteasome assays showed that gliotoxin had a minimal or no effect on the chymotrypsin like activity of the proteasome in CLL cells. In addition, the activity of the proteasome regulated transcription factor NFκB and the expression of its target genes like BCL2 and MCL1 were also not influenced by gliotoxin. These data point to the selectivity of targeting NOTCH2 signaling by gliotoxin rather than indirectly through the regulation of proteasome activity. Short term (4 hours) exposure of CLL cells revealed that NOTCH1 was equally transcribed in unstimulated and in PMA activated CLL cells. NOTCH2 was upregulated in PMA activated CLL lymphocytes whereas NOTCH4 was only weakly detectable in unstimulated CLL cells. Gliotoxin treatment resulted in the downregulation of NOTCH1, NOTCH2 and NOTCH4 mRNA expression. Interestingly, the inhibition of NOTCH2 activity by gliotoxin was associated with the concomitant induction of NOTCH3 signaling especially in the presence of PMA. This was indicated by the induced mRNA expression of NOTCH3 and its preferred target gene HEY1. Moreover, the induced transcription of HEY1 correlated with the upregulation of NR4A1, a key regulator of apoptosis in activated lymphocytes. These data may thus point to a pro-apoptotic role for NOTCH3/HEY1/NR4A1 signaling in CLL cells. The data also suggest that gliotoxin induced apoptosis is associated with differential regulation of the anti-apoptotic and pro-apoptotic arms of NOTCH signalling in CLL cells. RT-PCR revealed that NOTCH1 and NOTCH2 are the main NOTCH family members which are expressed in CLL cells under co-culture conditions with BMSC and in freshly isolated CLL cells. Exposure to gliotoxin in co-culture selectively induced apoptosis in CLL cells and led to downregulation of NOTCH1 and NOTCH2 together with upregulation of NOTCH3 mRNA expression. In summary, the data suggest that nuclear NOTCH2 activity might protect activated CLL cells from apoptosis by modulating the expression of NR4A1. The induced expression of NOTCH3 and its target gene HEY1 by gliotoxin reveals the complex role of different NOTCH family members in the regulation of apoptosis in CLL cells. Therefore, the individual NOTCH receptors may have opposite effects on CLL cell viability which should be considered in therapeutic approaches aimed to target NOTCH signaling in CLL. Disclosures: No relevant conflicts of interest to declare.

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Vorinostat Induced Cellular Stress Disrupts the Balance Between p38 MAPK and Erk Pathways Leading to Apoptosis in WM Cells.

Blood ◽

10.1182/blood.v114.22.3740.3740 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3740-3740

Author(s):

Jenny Sun ◽

Hsiuyi Tseng ◽

Lian Xu ◽

Zachary Hunter ◽

Bryan Ciccarelli ◽

...

Keyword(s):

Histone Deacetylase ◽

P38 Mapk ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Mapk Pathway ◽

Cellular Stress ◽

Family Members ◽

A Genome ◽

Caspase 7 ◽

Induced Apoptosis

Abstract Abstract 3740 Poster Board III-676 Despite advances, Waldenstrom's Macroglobulinemia (WM) remains incurable, and novel agents are urgently needed. Histone deacetylase (HDACs) are involved in transcription regulation and signal transduction of cells through a genome wide alteration in histone modification and other proteins, leading to significant increase in cellular stress in tumor cells. We therefore examined the activity of Vorinostat, a histone deacetylase inhibitor (HDAC-I), and dissected its pro-apoptotic molecular pathways in WM cells. Vorinostat exhibited dose dependent killing of both primary, bone marrow derived WM cells, as well as BCWM.1 WM cells with an LD50 of 3.5 to 5uM using Annexin V and PI staining. Vorinostat induced apoptosis in WM cells through activation of specific caspases at different time points. Caspase 3, 6, 8, and 9 were activated after 24 hours of Vorinostat. Even though caspase 7 is downstream of caspase 3, we observed that caspase 7 was activated earlier, starting at 6 hours. We therefore hypothesized that the regulators of caspase 7 may be affected by Vorinostat at an earlier time point. Further investigation confirmed that there was significant down-regulation of inhibitor of apoptosis (IAP) family members, including c-IAP1, c-IAP2, XIAP and Livin after 12 hours of treatment with Vorinostat, and may elude to greater sensitivity for IAPs in modulating Caspase 7 versus Caspase 3 in WM cells. We also studied the stress pathways including Erk, JNK and P38 pathways in Vorinostat treated WM cells. Activated p38, phospho-p38, was upregulated starting at 12 hours, while phopho-Erk abruptly decreased following 24 hours of treatment with Vorinostat. There was minimal change in the activity of JNK pathway following Vorinostat treatment. The activation of the p38 pathway coincided with a reduction in c-IAP1, c-IAP2, XIAP and Livin following treatment of WM cells with Vorinostat for 12 hours. Taken together, these studies support that stress induced apoptosis in WM cells is mediated through disruption in the balanced activity between the Erk and p38 MAPK pathways. Vorinostat induced cellular stress results in the activation of p38 MAPK pathway and a reduction of the IAP family members, leading to early activation of caspase 7. While the inhibition of Erk pathway by Vorinostat results in delayed activation of caspase 3, 6, 8, and 9 at 24 hours, the collective signaling strength of p38 activation as well as inhibition of Erk likely determines the apoptotic fate WM cells upon Vorinostat treatment. Disclosures: No relevant conflicts of interest to declare.

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Reversal of Drug/TRAIL-Resistant B-NHL Cells to Apoptosis by the Combination of Rituximab (anti-CD20) and Either Mapatumumab or Lexatumumab

Blood ◽

10.1182/blood.v116.21.4931.4931 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4931-4931

Author(s):

Mario I. Vega ◽

Sara Huerta-Yepez ◽

Melisa Martinez-Paniagua ◽

Stavroula Baritaki ◽

Haiming Chen ◽

...

Keyword(s):

Clinical Trials ◽

Tumor Cells ◽

Neutralizing Antibodies ◽

Conflicts Of Interest ◽

Phase 1 ◽

Death Receptor ◽

Hematologic Malignancies ◽

Direct Role ◽

Induced Apoptosis ◽

Anti Cd20

Abstract Abstract 4931 Conventional treatments of non-Hodgkin's lymphoma (B-NHL) consist primarily of chemotherapy. Currently, rituximab is used alone or in combination with chemotherapy. However, there are subsets of patients who do not respond initially or develop resistance to further treatment. Therefore, there is an urgent need to develop other immunotherapies with less toxicities. At present, both TRAIL and agonist antibodies directed against TRAIL-R1 and -R2 have been explored for various cancer treatments in various phase 1 and phase 2 clinical trials. We have recently demonstrated that rituximab sensitizes TRAIL-resistant B-NHL cells to TRAIL-induced apoptosis. Sensitization was the result of rituximab-induced inhibition of the constitutively activated NF-κB pathway and downstream the DR5 transcription repressor Yin Yang 1 (YY1). The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated in cells transfected with YY1 siRNA and that became sensitive to TRAIL- apoptosis. Treatment with rituximab did not have any observed effects on the expression of DR4. Based on these findings, it was possible that rituximab-mediated sensitization to TRAIL may invoke either TRAIL-R1 (DR4) or TRAIL-R2 (DR5), or both; thus, this possibility is currently being examined by the use of either neutralizing antibodies against each death receptor or by the use of silencing RNA. Currently, clinical trials are being conducted with both mapatumumab (anti-TRAIL-R1,) and lexatumumab (anti-TRAIL-R2) against a variety of cancers. These agonist antibodies have been evaluated clinically as single agents and in combination with standard therapy in solid and hematologic malignancies. It is not clear whether tumors can develop resistance to agonism of either one or both death receptors and thus, may not respond to monotherapy alone. Combination therapies may be required and we have hypothesized that the combination treatment of rituximab and agonist antibodies may be complementary or synergistic. This hypothesis was based on our findings that rituximab inhibits survival pathways and downregulates anti-apoptotic gene products and, thus, significantly reducing the threshold of resistance. Thus, this rituximab-mediated effect will facilitate the direct cytotoxicity of the agonist death receptor antibodies. The present study investigated whether rituximab can sensitize TRAIL-resistant tumor cells by either agonist TRAIL-R1 or TRAIL-R2 antibodies To address this question, we have examined the effect of agonist antibodies directed against either TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumamab). Treatment of the TRAIL-resistant Ramos B-NHL cells with rituximab for 24h and followed with treatment with non-toxic concentrations of mapatumumab (12 μg/ml) or lexatumumab (12 μg/ml) for 18h resulted in significant sensitization to apoptosis as assessed by activation of caspase 3. The mechanism of the sensitization by rituximab for each antibody was also examined. These findings demonstrated that rituximab sensitizes tumor cells to apoptosis by activation of either DR4 or DR5. Although there is heterogeneous expression of TRAIL-R1 and TRAIL-R2 in B-NHL cells, such cells may still be sensitive to rituximab-mediated sensitization to apoptosis by the corresponding agonist death receptor antibody. Recent findings demonstrated that some tumors expressing both DR4 and DR5 were shown to respond to TRAIL by preferential activation of DR4 and not DR5. Therefore, preclinical findings obtained with the use of TRAIL may not be predictive of outcome compared to the use of TRAIL-receptor specific agonist antibodies; mapatumumab or lexatumumab. Disclosures: No relevant conflicts of interest to declare.

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The interplay between BCL2 family members in response to pharmacologically induced apoptosis

10.1349/ddlp.1642 ◽

2010 ◽

Author(s):

Ting-Lan Tina Chang. Albershardt

Keyword(s):

Family Members ◽

Bcl2 Family ◽

Induced Apoptosis

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Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis

Tumor Biology ◽

10.1177/1010428317695016 ◽

2017 ◽

Vol 39 (3) ◽

pp. 101042831769501 ◽

Cited By ~ 3

Author(s):

Tea Soini ◽

Marjut Pihlajoki ◽

Antti Kyrönlahti ◽

Leif C Andersson ◽

David B Wilson ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Family Members ◽

B Cell Lymphoma ◽

Doxorubicin Treatment ◽

Bcl2 Family ◽

Apoptotic Effect ◽

High Doses ◽

Induced Apoptosis

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.

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Targeting Bcl-2 Proteins in Myeloma

Blood ◽

10.1182/blood-2019-121103 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. SCI-11-SCI-11

Author(s):

Martine Amiot

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Phase 1 ◽

Molecular Heterogeneity ◽

Malignant Cells ◽

Bh3 Mimetics ◽

Bcl2 Family ◽

Apoptotic Proteins ◽

Bh3 Mimetic

Escape from apoptosis is a hallmark of malignant cells that constitute an important mechanism associated to therapeutic resistance. Hematologic malignant cells are described as primed for apoptosis, a state in which pro-survival BCL2 family proteins sequester elevated levels of pro-apoptotic BH3-only proteins or even pre-activated BAX/BAK to ensure survival. Then, mimicking BH3-only proteins might preferentially turn on apoptosis in malignant cells. The understanding of BCL2 biology combined to drug discovery efforts have succeeded to the development of BH3-mimetics that bind with high affinity to a specific anti-apoptotic protein, promoting the release of pro-apoptotic members, leading to BAX and/or BAK activation and apoptosis. Venetoclax (ABT-199) is the first-in-class oral BH3-mimetic targeting selectively BCL2 and approved by the FDA for the treatment of CLL patients with 17p deletion. More recently, BH3-mimetics targeting MCL1 have been also described and are currently undergoing early phase clinical evaluation. Multiple myeloma (MM) displays a molecular heterogeneity, which includes hyperdiploid patients and patients with an Ig translocation with different chromosomes (4, 11, 6 or 16) leading to an over-expression of MMSET, CCND1, CCND3 or MAF genes, respectively. Analysis of apoptotic machinery diversity supports the notion that MM heterogeneity is extended to the BCL2 family content. The combined profile of BCL2, BCLXL and MCL1 mRNA expression in the main MM molecular subgroups was sufficient to discriminate each of them, suggesting that the composition of anti-apoptotic proteins should be taken into account for targeted therapies. MCL1 is over-expressed in MM and its strongest expression was found in the MMSET and MAF subgroups associated with an adverse prognosis. In contrast, the CCND1 subgroup expresses high levels of BCL2 and low level of MCL1 and BCLXL. In accordance to the heterogeneity of BCL2 member expression, different approaches such as MCL1 gene editing, BH3 profiling and in vitro sensitivity to specific BH3-mimetic demonstrate that myeloma cells are dependent on BCL2, MCL1 or BCLXL or co-dependent on two or three anti-apoptotic proteins. A plasticity of myeloma cell dependency was also reported according to disease status. Preclinical evaluation of venetoclax reveals that the sensitivity to venetoclax was mainly found in the CCND1 subgroup but not exclusively. The ratio of BCL2/BCLXL appears as a predictor of venetoclax sensitivity. A phase 1 clinical trial of venetoclax in relapsed/refractory myeloma patients demonstrate its efficacy mainly in t(11;14) patients with 40% of overall response. These results validate that targeting BCL2 as a very promising strategy for some patients, nevertheless stratification of patients based on t(11 ;14) and/or BCL2/BCLXL ratio is not sufficient to predict venetoclax response. Recent findings suggest that ex vivo pretreatment sensitivity could predict the clinical response to venetoclax. The reliability of the different approaches used to determine BCL2 dependency will be further discussed. A better comprehension of how to increase BCL2 priming is one of the most interesting perspectives to find the best combination regimens with venetoclax. Indeed, dexamethasone increases the BH3-only Bim protein and shifts it's binding towards BCL2. Phase 1 study of the combination of dexamethasone/venetoclax confirms the interest of this combination for t(11;14) patients. Finally, the possibility to overcome the resistance to venetoclax by targeting directly or indirectly the BCXL and MCL1 resistant factors could be of particular relevance. In conclusion, how basic research should work hand-in hand with clinical practice to design pertinent protocol to evaluate BCL2 targeted therapy will be examined. Disclosures No relevant conflicts of interest to declare.

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543 The high expression of pro-apoptotic BCL2 family members in uveal melanomas contribute to their sensitivity to MCL1 inhibitors

Journal of Investigative Dermatology ◽

10.1016/j.jid.2021.02.569 ◽

2021 ◽

Vol 141 (5) ◽

pp. S94

Author(s):

N. Mukherjee ◽

C. Dart ◽

C. Amato ◽

J. Skees ◽

A. Honig-Frand ◽

...

Keyword(s):

Family Members ◽

High Expression ◽

Bcl2 Family ◽

Uveal Melanomas

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Abstract CT210: Trial in Process: Phase 1 studies of BI 1701963, a SOS1::KRAS Inhibitor, in combination with MEK inhibitors, irreversible KRASG12C inhibitors or irinotecan.

10.1158/1538-7445.am2021-ct210 ◽

2021 ◽

Author(s):

Marco H. Hofmann ◽

Hengyu Lu ◽

Ulrich Duenzinger ◽

Daniel Gerlach ◽

Francesca Trapani ◽

...

Keyword(s):

Phase 1 ◽

Mek Inhibitors

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Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.1731.1731 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1731-1731

Author(s):

Mercè de Frias ◽

Daniel Iglesias-Serret ◽

Ana M Cosialls ◽

Llorenç Coll-Mulet ◽

Antonio F Santidrián ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Therapeutic Option ◽

Mrna Level ◽

Lymphocytic Leukemia ◽

Mrna Levels ◽

Dependent Manner ◽

Healthy Donors ◽

Induced Apoptosis

Abstract Abstract 1731 Poster Board I-757 Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL. Disclosures No relevant conflicts of interest to declare.

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