targeting akt elicits tumor suppressive functions of foxo ...multiple myeloma (mm) is a malignancy...

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Targeting AKT elicits tumor suppressive functions of FOXO transcription factors

and GSK3 kinase in Multiple Myeloma

Short title: FOXO and GSK3 act as tumor suppressors in MM

Timon A. Bloedjes1, Guus de Wilde1, Chiel Maas1, Eric E. Eldering2, Richard J. Bende1

Carel J.M. van Noesel1, Steven T. Pals1, Marcel Spaargaren1, and Jeroen E.J. Guikema1#

1Amsterdam UMC, University of Amsterdam, department of Pathology, Lymphoma and

Myeloma Center Amsterdam (LYMMCARE), The Netherlands; 2Amsterdam UMC,

University of Amsterdam, department of Experimental Immunology, Lymphoma and

Myeloma Center Amsterdam (LYMMCARE), The Netherlands.

Abstract word count: 229 (max 250 words)

Text word count: 3971 (max 4,000 words)

#Corresponding author: Jeroen E.J. Guikema, Ph.D. Amsterdam UMC, University of

Amsterdam, department of Pathology, Meibergdreef 9, 1105 AZ, Amsterdam, The

Netherlands, e-mail: [email protected], phone: +31-20-5665708, fax: +31-

20-5669523

All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/816694doi: bioRxiv preprint

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ABSTRACT

The phosphatidylinositide-3 kinases (PI3K) and the downstream mediator AKT drive

survival and proliferation of multiple myeloma (MM) cells and several AKT inhibitors are

currently being tested in clinical trials for MM patients. AKT inhibition has pleiotropic

effects, and the key aspects that determine therapeutic efficacy are not fully clear.

Therefore, we investigated the antimyeloma mechanism(s) of AKT inhibition. Among the

various downstream AKT targets are Forkhead box O (FOXO) transcription factors, and

we demonstrate that they are crucial for changes in gene expression upon AKT inhibition.

Based on gene expression profiling we defined an AKT-induced FOXO-dependent gene

set that has prognostic significance in a large cohort of MM patients, where low FOXO

activity correlates with inferior survival. We show that cell cycle exit and cell death of MM

cells after AKT inhibition required FOXO. In addition, glycogen synthase kinase 3 (GSK3),

a negatively regulated AKT substrate, proved to be pivotal to induce cell death and to

inhibit cell cycle progression after AKT inhibition. Finally, we demonstrate that FOXO and

GSK3 induced cell death by increasing the turnover of the myeloid cell leukemia 1 (MCL1)

protein. In concordance, the AKT inhibitor MK2206 greatly sensitized MM cells for the

MCL1 inhibitor S63845. Thus, our results indicate that FOXO and GSK3 are crucial

mediators of the antimyeloma effects of AKT inhibition, and suggest combination

therapies that may have therapeutic potential in MM.

KEYPOINTS

FOXO transcription factors and the GSK3 kinase are pivotal tumor suppressors downstream of AKT inhibition in MM cells.

FOXO and GSK3 activation after AKT inhibition leads to a decrease in MCL1 levels in MM cells resulting in cell death.


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INTRODUCTION

Multiple Myeloma (MM) is a malignancy of transformed clonal plasma cells that typically

reside in the bone marrow. Despite considerable improvements in the median survival

due to novel treatment modalities, patients inevitably relapse and become refractory to

further treatment. Further understanding of MM and plasma cell biology is urgently

needed and may lead to novel therapeutic strategies1.

The serine/threonine kinase AKT is a central node in the PI3K/AKT/mammalian target of

rapamycin (mTOR) pathway, which is active in MM due to growth factors produced by the

bone marrow microenvironment, or MM cells2–5. Furthermore, hemizygous deletions of

phosphatase and tensin homolog (PTEN), a negative regulator of AKT, were reported in

5-20% of MM patients and human myeloma cell lines (HMCL)6,7. AKT signaling is involved

in cell proliferation, survival and metabolism3,8. As such, it drives proliferation and sustains

the increased energy requirement of MM cells by reprogramming various metabolic

pathways8. Due to its crucial role in oncogenesis and cell survival, AKT is an attractive

therapeutic target for various types of cancer including MM, and consequently, several

clinical trials assessing the efficacy of AKT inhibitors in MM are ongoing9.

AKT has many substrates and pleiotropic effects in healthy and malignant cells. In

addition to metabolic, translational and mitogen-activated protein kinase (MAPK)

pathways8, forkhead box O transcription factors (FOXOs) and glycogen synthase kinase

3 (GSK3) are negatively regulated by AKT through phosphorylation8. The FOXOs , i.e.

FOXO1, FOXO3, FOXO4 and FOXO6, are context-dependent transcription factors that

act as tumor suppressors, but may also contribute to tumorigenesis10. Moreover, FOXO1

and FOXO3 have crucial and nonredundant functions in B-cell development, activation

and differentiation11–17. FOXOs can be phosphorylated, acetylated and ubiquitinated by


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a wide range of enzymes, thereby regulating their stability, localization and activity18.

Different interaction partners can also influence the specificity by which FOXO targets

genes, regulating their expression19. AKT phosphorylates GSK3 on Ser9 (beta-isoform)

and Ser21 (alpha-isoform), thereby inhibiting kinase activity20–22. GSK3 is a major AKT

target involved in the regulation of cell death by controlling BCL2-family proteins8,23–26.

In light of the recent interest in AKT as a therapeutic target in MM, we set out to provide

key insight into the antimyeloma mechanism(s) of AKT inhibition among its various

downstream pathways. Here, we demonstrate that FOXO1/3 and GSK3 are AKT-

restrained tumor suppressors, and that the expression of FOXO-dependent genes has

prognostic value in a cohort of MM patients. Mechanistically, we provide evidence that the

activation of FOXO and GSK3 provoked cell death in a nonredundant fashion through

negative regulation of MCL1, a major anti-apoptotic protein in plasma cells and MM26–28.

In accordance, AKT inhibition greatly sensitized MM cells for the MCL1 BH3-mimetic

S63845, even in MM cells resistant to AKT inhibition alone.

Our results show that the antimyeloma effects of AKT inhibition hinges on the activation

of FOXO1/3 and GSK3 and provide a clear rationale to explore combination therapies

aimed at AKT and its downstream targets, such as MCL1.


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MATERIALS AND METHODS

Cell culture and reagents

The human MM cell lines (HMCL) LME-1, MM1.S, XG-1, XG-3, LP-1, OPM-2, ANBL-6,

UM-3, and RPMI-8226 were cultured in Iscove’s modified Dulbecco’s medium (IMDM;

Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM of L-glutamine, 100

U/ml penicillin, 100 μg/ml streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA)

and 10% fetal calf serum (FCS; Hyclone, GE Healthcare Life Sciences, Pittsburgh, PA).

The cell lines XG-1, XG-3 and ANBL-6 were cultured in medium supplemented with 1

ng/ml interleukin-6 (IL-6; Prospec Inc, Rehovot, Israel), which was washed out prior to

experiments. HEK293T cells were obtained from the American Type Culture Collection

(ATCC, Manassas, VA) and cultured in supplemented Dulbecco's modified eagle medium

(DMEM; Invitrogen Life Technologies) and 10% FCS. The following small-molecule

inhibitors were used: GSK2110813 (Afuresertib) (AKT inhibitor; Selleckchem, Houston,

TX) MK2206 (AKT inhibitor; Selleckchem), CHIR99021 (GSK3 inhibitor, Sigma Aldrich,

St. Louis, MO), AS1842856 (FOXO1 inhibitor, Merck, Darmstadt, Germany), S63845

(MCL1 inhibitor, Selleckchem), cycloheximide (Sigma Aldrich).

Constructs and retroviral/lentiviral transductions

CRISPR/Cas9 knockout (KO) HMCL clones were generated by lentiviral transduction as

described previously29. HMCLs overexpressing MCL1 were generated by retroviral

transduction using the LZRS-MCL1-IRES-GFP plasmid. A more detailed description is

available in the supplemental materials and methods.

Patient samples


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Primary tumor cells from MM patients (>80% plasma cells) were enriched by Ficoll-paque

PLUS (GE Healthcare Life Sciences) density centrifugation. Patient material was

obtained according to the ethical standards of our institutional medical ethical committee,

as well as in agreement with the Helsinki Declaration of 1975, as revised in 1983. Primary

MM cells were cultured overnight in IMDM + 10% FCS, supplemented with 1 ng/ml IL-6

before being used in further experiments.

Immunoblotting

Immunoblotting experiments were performed as described previously30. Protocols and

antibodies used are available in the supplemental materials and methods, densitometry

quantification of immunoblots was performed using Image J software (imagej.net)31.

Gene expression profiling

RNA from 2 x 106 cells was isolated using TRI-reagent (Sigma-Aldrich) and purified using

the RNEASY mini kit (Qiagen, Hilden, Germany) using the RNA cleanup protocol supplied

by the manufacturer. The RNA was analyzed using Affymetrix Human Genome U133

Plus 2.0 arrays (Affymetrix, Santa Clara, CA) and normalized using MAS5.0 (accession

no. GSE120941). Gene expression data was analyzed using the R2: Genomics Analysis

and Visualization Platform (http://r2.amc.nl). Venn diagrams were prepared using

BioVenn (www.biovenn.nl)32. Enrichment plots were generated using the Broad Institute

gene set enrichment analysis (GSEA) computational method and software33.

Flow cytometry


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Specific cell death was assessed by 7-aminoactinomycin-D (7-AAD; Biolegend Inc, San

Diego, CA) staining and flow-cytometry and calculated as reported earlier34. Cell cycle

analysis was performed by determining DNA content and bromodeoxyuridine (BrdU)

incorporation as described previously29. A detailed protocol is provided in the

supplemental materials and methods.

Statistics

Statistical analysis was performed using the Graphpad Prism software package

(Graphpad Software, La Jolla, CA) and combination indexes were calculated using

CompuSyn (ComboSyn Inc, Paramus, NJ)35. In case single dose drug combinations were

used we calculated the expected effect of drug combinations (C) using the Bliss

independence model [C=A+B-(A*B)] where A and B indicate the observed cell death at

specific concentrations of the single drugs36.


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RESULTS

Inhibition of AKT induces cell death in MM cell lines and patient samples

To investigate the effects of AKT inhibition we exposed human MM cell lines (HMCL) (n=9)

to increasing concentrations of the allosteric AKT inhibitor MK2206 or the ATP competitive

AKT inhibitor GSK2110183 (Afuresertib), both pan-AKT (AKT1/2/3) inhibitors that are

currently being evaluated for clinical activity in MM and other cancer patients37,38. Both

inhibitors potently induced cell death in the majority of the HMCLs. However, UM-3 and

RPMI-8226 were refractory to AKT inhibitor-induced cell death (Fig 1A). Enriched

malignant plasma cells obtained from MM patients (n=6) (Suppl Fig 1A) showed a similar

response, in which MK2206 induced cell death to a variable degree in all but one patient

(Fig 1B).

To assess the downstream effects of AKT inhibition we performed immunoblotting for

several established AKT targets. In all HMCLs except RPMI-8226, MK2206 decreased

phosphorylation of AKT Thr308 and Ser473, targets of protein kinase PDK1 and mTOR

complex 2 (mTORC2), respectively, that regulate AKT kinase activity8. The

phosphorylation of downstream AKT substrates, FOXO1, FOXO3, GSK3beta and the

ribosomal protein S6 were clearly decreased, indicating that MK2206 effectively blocked

AKT function in all HMCLs except RPMI-8226. In agreement, similar effects of AKT

inhibition were observed in primary MM patient samples (Fig 1D).

These results confirm that FOXO and GSK3 are activated upon inhibition of AKT in the

context of HMCLs and primary MM cells.

FOXO transcription factors are required for AKT inhibitor-induced cell death of MM cells


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To assess whether FOXO transcription factors were required for AKT inhibitor-induced

cell death in MM cells we generated several FOXO1- and FOXO3-knockout clones for

LME-1 (n=2), MM1.S (n=4) and XG-3 (n=4) using CRISPR/Cas9. Loss of FOXO protein

expression was confirmed by immunoblotting (Fig 2A). The loss of FOXO had no

apparent effect on MM cell survival under basal condition (data not shown). However,

AKT inhibitor-induced cell death was nearly abrogated in FOXO1-deficient LME-1 cells,

whereas a small but significant reduction in cell death was observed in the FOXO3-

deficient LME-1 cells (Fig 2B, Suppl Fig 2A). In contrast, MK2206-induced and

GSK211083-induced cell death was almost abolished in the FOXO3-deficient MM1.S and

XG-3 cell lines, while FOXO1-deficient cells remained sensitive (Fig 2B, Suppl Fig 2A).

Of interest, we observed a slight increase in FOXO3 protein expression in FOXO1-

deficient LME-1 cells, and a substantial increase in FOXO1 expression in FOXO3-

deficient XG-3 cells (Fig 2A).

We confirmed the tumor suppressive function of FOXO in MM using AS1842856, a small

molecule inhibitor that blocks the transcriptional activity of FOXO1 and to a far lesser

extent that of FOXO339. In line with results observed in the FOXO-deficient cells,

AS1842856 rescued LME-1 cells after MK2206 treatment but had almost no effect on the

induced cell death in MM1.S cells. In other HMCLs, AS1842856 varyingly rescued

MK2206-induced cell death (Fig 2C). These results were confirmed in MM patient

samples, where AS1842856 significantly inhibited MK2206-induced cell death in 4 out of

5 patient samples tested (Fig 2D). The varying degree of rescue from cell death by

AS1842856 may reflect the differential dependency on FOXO1 versus FOXO3 in these

patients. Similarly, AS1842856 had no additional effect on FOXO1-deficient LME-1 cells,

whereas AKT inhibitor-induced cell death of MM1.S cells, which required FOXO3, was


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partially rescued by AS1842856 (Suppl Fig 2B). These results clearly demonstrate that

FOXO transcription factors are crucial effectors of cell death upon inhibition of AKT, and

function as AKT-restrained tumor suppressors in MM.

Inhibition of AKT activates FOXO-controlled transcriptional regulation in MM cells

To determine FOXO-dependent transcriptional changes we performed gene expression

profiling (GEP) of two independently established FOXO1-knockout clones from the LME-

1 HMCL, and of two FOXO3-knockout clones from the MM1.S and XG-3 HMCLs,

respectively. Cloned wildtype “Cas9 only” cells (WT) were used as controls. We

anticipated that the three HMCLs would show considerable variance in the expression of

FOXO target genes, due to the heterogeneous genetic backgrounds of these HMCLs.

However, gene set enrichment analysis (GSEA) on the combined GEP datasets clearly

indicated significant enrichment of a FOXO3 target gene set in the AKT inhibitor-treated

WT control clones ('WTMK'; WT clones, MK2206-treated) versus the untreated control

and FOXO-knockout clones, and AKT inhibitor-treated FOXO-knockout clones ('REST')

(Fig 3A).

Using variable cutoffs, we identified FOXO-regulated genes in MM1.S (848 up, 1541

down), XG-3 (329 up, 382 down) and LME-1 (438 up, 457 down) (Suppl Table 1). In

agreement, k-means unsupervised learning and principal component analysis (PCA) for

MM1.S and XG3 showed that the MK2206-treated control clones ('WTMK') consistently

clustered together versus a cluster consisting of the untreated control and FOXO3-

knockout clones, and the MK2206-treated FOXO3-knockout clones ('REST'). In contrast,

the MK2206-treated control and FOXO1-deficient clones clustered together versus the

untreated clones in LME-1. This may reflect intrinsic differences between LME-1 versus


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MM1.S and XG-3, and/or may indicate that FOXO1 has a less pronounced effect on gene

expression compared to FOXO3 (Suppl Fig 2A, B). Comparing these datasets, 23 genes

were found to be consistently upregulated and 44 genes were downregulated upon AKT

inhibition in a FOXO-dependent fashion (Fig 3B), among which are the established direct

FOXO targets CITED2 (found in all three HMCLs) and PIK3CA (found in LME-1 and XG-

3)40,41 (Suppl Table 1). Despite this overlap, the majority of FOXO-dependent genes

were specific for the different HMCLs, underscoring the heterogeneous and context-

dependent nature of the transcriptional consequences of FOXO activation.

The FOXO-dependent downregulated genes found in all three HMCLs

(FOXO_shared_down) were used to perform a k-means unsupervised learning analysis

(2 groups, 10 rounds) on a MM patients GEP dataset (n=542) that includes clinical data42.

Patients were clustered in 2 groups with respectively, low expression of FOXO

suppressed genes (signifying high FOXO activity) versus high expression of FOXO

suppressed genes (low FOXO activity) (Fig 3C). Importantly, patients with high

expression of FOXO suppressed genes, reflecting high AKT activity, show an inferior

overall survival (p=0.000028) (Fig 3D). The 90% survival was 9 months in this group with

low FOXO activity versus 25 months in the high FOXO activity group, and the 2-year

survival was 75% versus 91%. These results show that the loss of FOXO activity (and/or

increased AKT activity) results in a more aggressive disease course, consistent with a

tumor suppressive role of FOXO in MM.

Inhibition of AKT induces a FOXO-dependent cell cycle arrest in HMCLs

Further inspection of the GSEA data on the combined datasets showed significant

depletion of cell cycle and DNA replication/repair-associated gene sets in the MK2206-


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treated control clones (Fig 4A, Supplemental Fig 4A), indicating that activation of FOXO

induced cell cycle exit. In agreement, MM patients clustered according to high FOXO

activity showed a significant depletion of these gene sets (Suppl Fig 4B).

Correspondingly, cell cycle analysis showed that MK2206 treatment resulted in a

significant loss of S phase and a concomitant increase in G1 phase in 7 out of 8 HMCLs

tested (Fig 4B), including the UM-3 HMCL that was unresponsive to AKT inhibition

regarding cell death (Fig 1A). Cell cycle exit was dependent on FOXO1 in LME-1 and on

FOXO3 in MM1.S and XG-3 (Fig 4C). Expression of the cyclin-dependent kinase 4

(CDK4) protein, which regulates G1 phase progression, was diminished by AKT inhibition

in a dose-dependent manner in MM1.S and XG-3 control cells but not in FOXO3-deficient

cells. In contrast, CDK4 remained largely unaffected in LME-1 cells. Protein expression

of C-MYC was reduced in all three cell lines in a FOXO-dependent manner, which is also

reflected in the GSEA analysis of the MYC targets geneset (Fig 4A). Furthermore, C-

MYC displayed higher basal protein levels in the FOXO1-deficient LME-1 cells and

FOXO3-deficient XG-3 cells compared to control cells. Protein expression of Cyclin D2

was down modulated after AKT inhibition in a FOXO3-dependent manner in MM1.S and

XG-3, but appeared to be activated in the FOXO1-deficient LME-1 cells (Fig 4D). Analysis

of GEP data indicated that the levels of CDK4 mRNA were consistently down modulated

by FOXO3-activation in MM1.S and XG-3, but not in LME-1 cells, suggesting that CDK4

gene transcription is suppressed by FOXO3, but not FOXO1. In contrast, C-MYC mRNA

levels were not affected in any of the HMCLs, whereas CCND2 mRNA levels showed a

FOXO3-dependent decrease in expression after AKT inhibition in XG-3 and MM1.S

(Suppl Fig 5). These data indicate that the effects of FOXO activation on the cell cycle


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involves both transcriptional and post-transcriptional mechanisms, which are context-

dependent, but nonetheless result in a uniform cell cycle exit.

GSK3 kinase activity is involved in AKT inhibitor-induced cell death and cell cycle arrest

GSK3 is an important physiological target of AKT that is inhibited by phosphorylation (Fig

1C, D)20. Activation of GSK3 downstream of AKT and PI3K inhibition has been implicated

in apoptosis and cell cycle arrest21,43. Correspondingly, the GSK3-specific kinase inhibitor

CHIR99021 significantly diminished cell death of AKT inhibitor-treated MM cells. Inhibition

of GSK3 resulted in a partial rescue of MK2206-induced cell death, ranging from a 1.4-

fold decrease in MM1.S cells, to a 3.4-fold decrease in LME-1 cells (Fig 5A). A similar

range of decrease in AKT inhibitor-induced cell death was observed in primary MM patient

plasma cells co-treated with CHIR99021 (Fig 5B). GSK3 inhibition prevented AKT

inhibitor-induced cell cycle arrest in the LP-1 and LME-1 HMCLs, whereas it had a modest

effect in XG-3 and MM1.S. The effects of AKT inhibitor and GSK3 inhibitor treatment in

XG-1 displayed a similar trend on the cell cycle as LME-1 and LP-1 but did not reach

significance (Fig 5C). Of note, in LME-1, LP-1 and XG-3 cells we observed a significant

increase in S phase upon treatment with the GSK3 inhibitor alone, suggesting that

constitutive AKT signaling in these HMCLs does not completely impede GSK3 kinase

activity. The activation of FOXO1 or FOXO3 appeared not to be abrogated by GSK3

inhibition (Fig 5D). These results indicate that GSK3 significantly contributed to the

antimyeloma properties of AKT inhibition, acting in a cooperative fashion with FOXO

transcription factors.


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GSK3 and FOXO activation upon AKT inhibition results in decreased MCL1 expression

and sensitizes HMCLs for a selective MCL1 BH3 mimetic

Previously, it was shown that AKT and GSK3 kinase activity are involved in apoptosis by

regulating the protein stability of MCL126,44,45, a BCL2-family member that is essential for

the survival of non-malignant plasma cells and myeloma cells27,46. These findings

prompted us to investigate the effects of AKT inhibition on MCL1 protein levels in MM.

MCL1 protein expression was diminished by AKT inhibitor treatment in responsive HMCLs

(LME-1, MM1.S and XG-3), which depended on FOXO and GSK3 activity (Fig 6A).

Similarly, MCL1 protein expression was decreased in AKT inhibitor-treated primary MM

patient plasma cells (Fig 6B). Cycloheximide chase experiments showed that AKT

inhibitor treatment increased MCL1 protein turnover in responsive HMCLs (Fig 6C). In

contrast, BCL2 and BCL-XL protein stability remained unchanged after AKT inhibition in

all tested HMCLs (Suppl Fig 6A). In agreement, overexpression of MCL1 in LME-1,

MM1.S and XG-3 prevented AKT inhibitor-induced cell death (Fig 6D, E). The S63845

small molecule inhibits MCL1 and displays potent antimyeloma acitivity47. Based on our

results we asked whether AKT inhibition sensitized myeloma cells for this MCL1 inhibitor.

We exposed the MK2206-responsive HMCLs LME-1, MM1.S, XG-3 and the unresponsive

HMCLs UM-3 and RPMI-8226 to increasing concentrations of MK2206, S63845 and the

combination of both. A clear potentiating effect on induced cell death was observed with

the combination of inhibitors, even in the MK2206 unresponsive HMCLs (Fig 6F, Suppl

Fig 6B).

In accordance with the observed results in HMCLs, the combination of AKT and MCL1

inhibitors resulted in cell death consistently higher than the predicted Bliss score in primary

MM cells, indicating a potentiating effect of this drug combination (Fig 6G). These results


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indicate that the FOXO- and GSK3-mediated decrease in MCL1 protein expression after

AKT inhibition sensitizes myeloma cells for the MCL1-specific inhibitor S63845, improving

the efficacy of these novel therapeutic modalities.


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DISCUSSION

Here we showed that the FOXO1 and FOXO3 transcription factors and the GSK3 kinase

act as AKT-repressed tumor suppressors in MM cells. As such, FOXO1/3 and GSK3 are

critical mediators of the antimyeloma effects of AKT-targeted therapy. In agreement, we

showed that the expression levels of a set of FOXO targets genes is related to overall

survival rates in MM patients; low FOXO activity (reflecting high AKT activity) identifies a

patient subgroup with inferior survival.

We demonstrated that upon AKT inhibition FOXO1/3 and GSK3 mediate cell cycle exit by

repressing genes involved in DNA replication and cell cycle progression, and cause cell

death by provoking the loss of MCL1 protein expression. These observations offer

important leads to improve therapeutic strategies aimed at the PI3K/AKT pathway. As an

example, we showed that the AKT inhibitor MK2206 synergized with the recently

developed MCL1 inhibitor S6384547. Combination of these two drugs very efficiently

caused cell death of MM cells, even in cells refractory to AKT inhibition, warranting further

investigation into the clinical efficacy of such combination therapies. Targeting AKT and

MCL1 simultaneously can be considered a vertical inhibition strategy, in which two points

of the same pathway are inhibited. Similar vertical inhibition strategies for the

PI3K/AKT/mTOR pathway have shown to be synergistic in multiple cancer types48–50.

The tumor suppressive roles of FOXO1/3 and GSK3 partly explain the constitutive

activation of the PI3K/AKT pathway in MM cells, underscoring its crucial function in tumor

cell survival. Whether PI3K/AKT signaling has a similar role in the maintenance of normal

plasma cells remains unknown. However, AKT activity was shown to be important in the

development of normal plasma cells, as in vitro differentiation of mouse plasma cells was

inhibited by forced expression of constitutive active FOXO1. Conversely, FOXO1


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knockout in mouse mature B cells or treatment with PI3K inhibitors increased plasma cell

formation12,51. In contrast, expression of constitutive active FOXO1 in classical Hodgkin

lymphoma directly drove PRDM1/BLIMP1 expression, thereby activating a plasma cell

gene signature52. The role of FOXO3 in plasma cell differentiation is less clear, whereas

FOXO3-deficient mouse mature B cells show no apparent defects in plasma cell

development13. However, expression of FOXO3 increases from germinal center B cells

to plasma cells53. These observations suggest that FOXO transcription factors act in a

context-dependent fashion in normal and malignant plasma cells. This is emphasized by

our GEP data, showing relatively limited overlap between FOXO-regulated genes in three

different HMCLs. Despite this apparent heterogeneity, the effects of FOXO activation on

proliferation and cell death were remarkably uniform, as reflected by gene expression

enrichment analysis performed on the combined datasets. There are some reports that

suggest that FOXO1 and FOXO3 are functionally linked and act redundantly, for instance

in autophagy54 and development of thymic lymphomas and hemangiomas55, whereas in

lymphocyte development these FOXO transcription factors have specialized as well as

redundant functions12,13. However, in MM cells the functions of FOXO1 and FOXO3

suggest there is no obvious overlap, since the loss of FOXO1 was not compensated by

FOXO3, or vice versa, despite increased expression of the alternate family member.

A major difference between normal and malignant plasma cells is their proliferative

capacity, which can be attributed to recurrent genomic abnormalities that result in the

aberrant expression of cell cycle related genes, such as D-type cyclins and C-MYC, which

are nearly universal events in MM56. Despite aberrant expression, these cell cycle-

associated genes were nevertheless repressed by FOXO1/3 upon AKT inhibition, thereby

reversing the oncogenic proliferative program of MM cells. Pharmacological inhibition of


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AKT resulted in a FOXO-dependent G1 phase arrest in MM cells, consistent with earlier

reports on lymphomas and other cancer cell types57–60. As previously shown, FOXO may

cause cell cycle arrest by driving the expression of the cyclin-dependent kinase inhibitor

p27(kip1)61, and by reducing the expression of D-type cyclins62. We found that AKT

inhibition resulted in a FOXO-dependent down modulation of cyclin D2, CDK4 and C-MYC

protein expression, whereas p27(kip1) was not affected (data not shown). In agreement,

GSEA indicated that MYC target genes were significantly depleted from AKT inhibitor-

treated control cells (Suppl Fig 4). In addition, DNA repair gene expression signatures

were significantly down modulated upon activation of FOXO1/3 in MM (Suppl Fig 4),

suggesting that combining AKT inhibitors with DNA-damaging agents might be a

promising treatment option for MM patients.

Our date emphasizes the pivotal importance of FOXO transcription factors and GSK3

kinase activation on MM cell survival and cell cycle progression downstream of AKT

inhibition. AKT-mediated phosphorylation of FOXO1 or FOXO3 was not affected by GSK3

kinase inhibition, suggesting that GSK3 did not act upstream of FOXO1/3. These data

indicate that FOXO1/3 and GSK3 act in a cooperative fashion. The role of GSK3 in the

regulation of cell death downstream of PI3K/AKT signaling was described previously in

various types of cancer21,63–66. However, the role of GSK3 in MM is less clear, as both

prosurvival and proapoptotic functions have been ascribed to this kinase67–73. To our

knowledge, our results are the first to indicate that GSK3 is an important mediator of cell

death in MM cells controlled by AKT signaling. The partial and heterogeneous effect of

GSK3 inhibition most likely reflects the molecular heterogeneity of the HMCLs and patient

samples used in this study.


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Recently, it has become clear that MM displays marked clonal heterogeneity, and that the

tumor consist of subclonal variants that undergo clonal evolution during treatment and

progression. Moreover, the mutation spectrum also may change over time, alluding to

ongoing mutagenic processes that affect new candidate genes involved in therapy

resistance and disease progression74,75. Based on our data, it is conceivable that

treatment aimed at the PI3K/AKT pathway in MM may result in the selection, or

appearance, of subclonal variants that harbor mutations inactivating FOXO and/or GSK3.

Currently, several clinical trials assessing the efficacy of AKT inhibitors for the treatment

of MM are underway. Our data underscores that inhibition of AKT can induce tumor cell

vulnerabilities that can be exploited therapeutically, such as the AKT mediated negative

regulation of MCL1.


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73. Qiang Y-W, Ye S, Chen Y, et al. MAF protein mediates innate resistance to proteasome inhibition therapy in multiple myeloma. Blood. 2016;128(25):2919–2930. 74. Bolli N, Avet-Loiseau H, Wedge DC, et al. Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat. Commun. 2014;5:2997. 75. Corre J, Cleynen A, Robiou du Pont S, et al. Multiple myeloma clonal evolution in homogeneously treated patients. Leukemia. 2018;

ACKNOWLEDGEMENTS

This research was supported by the Netherlands Organization for Scientific Research

Innovational Research Incentives Scheme VIDI grant no.16126355, and by grant AMC

2018-11597 of the Dutch Cancer Society (both to J.E.J.G)

AUTHORSHIP AND CONFLICT-OF-INTEREST STATEMENTS

J.E.J.G. and T.A.B. designed the research; T.A.B., G.d.W., C.M. and J.E.J.G. performed

the experiments; E.E., R.J.B., C.J.v.N., S.T.P., M.S. and J.E.J.G. analyzed the data;

T.A.B., G.d.W. and J.E.J.G. wrote the manuscript; and all authors edited the manuscript.

E.E. received research funding from Hoffman-La Roche Ltd. and from Gilead Sciences

Inc.


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25

LEGENDS TO THE FIGURES

FIGURE 1. Inhibition of AKT in MM cells induces cell death

(A) Percent of specific cell death of HMCLs (n=9) treated with increasing concentrations

of the ATP-competitive AKT inhibitor GSK2110183 (Afuresertib) (left panel) and the

allosteric AKT inhibitor MK2206 (right panel) for 3 days. Specific cell death was calculated

based on 7-AAD viability dye staining and flow-cytometry. Mean values of 3 independent

experiments are shown. (B) Percent of specific cell death of primary MM plasma cells

from patients (n=6) treated with 2.5 M MK2206 AKT inhibitor for 3 days. Specific cell

death was calculated based on 7-AAD viability dye staining and flow-cytometry. Means ±

SEM of three technical replicates are displayed, n=3 (****p<0.0001; ***p<0.001; **p<0.01;

one sample t-test). (C) Immunoblot analysis of protein expression in AKT-inhibitor treated

HMCLs LME-1, MM1.S, XG-1, XG-3, ANBL-6, LP-1, OPM-2, UM-3 and RPMI-8226. Cells

were serum starved for one hour, after which they were incubated in medium containing

10% FCS with or without 2.5 M MK2206 for 2 hours. Shown are the indicated proteins,

-actin was used as a loading control. Representative immunoblot of at least 2

independent experiments is shown. (D) Immunoblot analysis of protein expression in

primary MM patient plasma cells (n=4) serum starved for one hour, after which they were

incubated in medium containing 10% FCS with or without 2.5 M MK2206 for 2 hours.

Shown are the indicated proteins, -actin was used as a loading control.


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FIGURE 2. Cell death induced by AKT inhibition is dependent on FOXO1 or FOXO3

in MM cells

(A) Immunoblot analysis of CRISPR-Cas9 generated FOXO1 and FOXO3 knockout

clones of the LME-1 (n=2), MM1.S (n=4), XG-3 (n=4) HMCLs. -actin was used as loading

control. (B) AKT inhibitor-induced cell death is dependent on the presence of FOXO1 in

LME-1, and on FOXO3 in MM1.S and XG-3. Cloned knockout and control HMCLs were

treated for 3 days with various concentrations of the MK2206 AKT inhibitor. 2 to 4

independently established clones were analyzed per condition. Red bars depict FOXO1

knockout clones, blue bars depict FOXO3 knockout clones. Means ± SEM of 3

independent experiments are shown (****p<0.0001; **p<0.01; ns = not significant; one-

way ANOVA with Dunnet’s multiple comparison test). (C) AKT inhibitor-induced cell death

in HMCLs can be rescued by FOXO1 inhibition (n=5). HMCLs were treated for 3 days

with 3.2 M MK2206 AKT inhibitor, with (grey bars) or without (black bars) 100 nM of the

FOXO1 inhibitor AS1842856. Means ± SEM of 3 independent experiments are shown

(****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns = not significant; unpaired t-test with

Welch’s correction). (D) Cell death of primary MM patient plasma cells induced by AKT

inhibitor MK2206 (2.5 M) can be overcome by the FOXO1 inhibitor AS1842856 (n=5).

Cells were treated for 3 days with 3.2 M MK2206 AKT inhibitor, with (grey bars) or without

(black bars) 100 nM of the FOXO1 inhibitor AS1842856. Means ± SEM of 3 technical

replicates are shown (****p<0.0001; ***p<0.001; ns = not significant; unpaired t-test with

Welch’s correction). Specific cell death in these experiments was determined by 7-AAD

viability dye staining and flow-cytometry.


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FIGURE 3. Inhibition of AKT induces a FOXO-dependent gene signature in MM cells

Independent LME-1 FOXO1 knockout clones (n=2), MM1.S FOXO3 knockout clones

(n=2) and XG-3 FOXO3 knockout clones and their respective control clones (n=2) were

treated overnight with 2.5 M MK2206 AKT inhibitor, or left untreated, and subjected to

gene expression profiling. (A) GSEA enrichment plot of upregulated FOXO3 target genes

(DELPUECH_FOXO3_TARGETS_UP) in wildtype “cas9 only” (WT) clones treated

overnight with 2.5 M MK2206 (‘WTMK’; left side of the plot) versus MK2206-treated

FOXO knockout clones, untreated WT and FOXO knockout clones (‘REST’; right side of

the plot) from the LME-1, MM1.S and XG-3 HMCLs combined. False discovery rate

(FDR), enrichment score (ES), normalized enrichment score (NES) and p-value are

indicated in the enrichment plot. (B) Area proportional Venn diagrams depicting the

number of genes that are upregulated (left panel) or downregulated (right panel) in a

FOXO-dependent fashion upon AKT inhibition. Genes that overlap in all 3 HMCLs are

listed alongside the Venn diagrams. Differentially expressed genes between the groups

were defined based on p-values, using the following cutoffs: LME-1 p<0.15, MM1.S

p<0.01, XG-3 p<0.02, (Annova corrected for multiple testing by false discovery rate). (C)

K-means clustering results (10 rounds, 2 groups, blue and red boxes) and z-score heat

maps based on the genes that are downregulated upon AKT inhibition in a FOXO-

dependent fashion and overlapped in all 3 HMCLs (see Fig 3B, right) in a patient GEP

dataset. This set contains gene expression profiling- and survival data of 542 MM

patients. Blue depicts downregulated gene expression and red depicts upregulated gene

expression. (D) Kaplan-Meier plot depicting overall survival of MM patients from the GEP


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28

dataset, using k-means clustering derived groups representing high and low expression

of FOXO target genes (see Fig 3B).

FIGURE 4. AKT inhibition impairs proliferation in a FOXO-dependent fashion in MM

cells

(A) GSEA enrichment plots show a significant depletion of cell cycle and proliferation

associated gene sets in wildtype “cas9 only” (WT) clones treated overnight with 2.5 M

MK2206 (‘WTMK’; left side of the plots) versus MK2206-treated FOXO knockout clones

and untreated WT and FOXO knockout clones (‘REST’; right side of the plots). For GSEA,

GEP datasets from the LME-1, MM1.S and XG-3 HMCLs were combined. False discovery

rate (FDR), enrichment score (ES), normalized enrichment score (NES) and p-values are

indicated in the enrichment plots. (B) BrdU incorporation cell cycle analysis of HMCLs

(n=8) treated overnight with 2.5 M MK2206. BrdU incorporation and DNA content was

assessed by flow-cytometry. Sub G1 phase (dead) cells were excluded from the analysis.

Percentages of cells in the G1, S, and G2 phase of the cell cycle are depicted. Statistical

analysis (one-way ANOVA with Fisher’s Least Significant Difference post-test) was

performed on the percentages of cells in S phase (***p<0.001; **p<0.01; *p<0.05; ns =

not significant). The mean values of three experiments are depicted. (C) AKT inhibition

leads to a FOXO-dependent G1 phase arrest. Cell cycle analysis of LME-1, MM1.S and

XG-3 HMCLs and their respective FOXO1 or FOXO3 knockout clones treated overnight

with 2.5 M MK2206 (MK). Percentages of cells in the G1, S, and G2 phase of the cell

cycle are depicted. Statistical analysis (one-way ANOVA with Bonferroni’s multiple

comparison test) was performed on the percentages of cells in S phase compared to


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29

untreated control clones (****p<0.0001; ***p<0.001; ns = not significant). The mean

values of 3 experiments are depicted. (D) Immunoblot analysis of cell cycle and

proliferation associated proteins in LME-1, MM1.S and XG-3 control clones and their

respective FOXO1 or FOXO3 knockout clones treated overnight with increasing

concentrations of 0/0.5/2.5 M MK2206. -actin was used as loading control.

FIGURE 5. Inhibition of GSK3 partially rescues MM cells from AKT inhibitor-

induced cell death and cell cycle arrest

(A) GSK3 inhibition partially rescued AKT inhibitor-induced cell death in HMCLs (n=5).

Various concentrations of MK2206 were used for the different HMCLs (LME-1, LP-1 and

XG-1: 3.2 M; XG-3: 0.4 M; MM1.S: 0.8 M). Cells were co-treated with 1 M

CHIR99021 (GSK3 inh.) for 3 days. Means ± SEM of 3 independent experiments are

shown (****p<0.0001; ***p<0.001; **p<0.01; unpaired t-test with Welch’s correction). (B)

Partial rescue of AKT inhibitor-induced cell death in primary MM patient plasma cells

(n=5). Cells were treated for 3 days with 2.5 M MK2206 and 1 M CHIR99021. Specific

cell death was determined by 7-AAD viability staining and flow-cytometry. Means ± SEM

of 3 technical replicates are shown (****p<0.0001; ***p<0.001; **p<0.01; unpaired t-test

with Welch’s correction). (C) BrdU incorporation cell cycle analysis of HMCLs (n=5)

treated overnight with 2.5 M MK2206 and 1 M CHIR99021. BrdU incorporation and

DNA content was assessed by flow-cytometry. Sub G1 phase (dead) cells were excluded

from the analysis. Percentages of cells in the G1, S, and G2 phase of the cell cycle are

depicted. Statistical analysis (one-way ANOVA with Bonferroni’s multiple comparison

test) was performed on the percentages of cells in S phase compared to untreated control


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30

cultures. Cultures were performed in triplicate (**p<0.01; *p<0.05; ns = not significant).

(D) Immunoblot analysis of phospho-Thr24 FOXO1/phospho-Thr32 FOXO3 in LME-1

cells and MM1.S cells treated overnight with 0, 0.5 or 2.5 M MK2206, with or without 1

M CHIR99021. -actin was used as loading control.

FIGURE 6. AKT inhibition in MM cells leads to the FOXO/GSK3-mediated MCL1

down modulation resulting in cell death

(A) Immunoblot analysis of MCL1 protein expression in wildtype “cas9 only” clones treated

overnight with increasing concentrations of MK2206 (0; 0.5; 2.5 M) with or without 1 M

CHIR99021, and in MK2206-treated (0; 0.5; 2.5 M) FOXO1 or FOXO3 knockout HMCLs.

(B) Immunoblot analysis of MCL1 protein expression in primary MM patient plasma cells

(n=5) treated overnight with 2.5 M MK2206. (C) Immunoblot analysis of MCL1 protein

stability in HMCLs (n=5) after cycloheximide (CHX) treatment (200 µg/ml), with or without

pretreatment of 2.5 µM MK2206 for 12 hours. Cells were treated with CHX as indicated

by depicted time points. (D) Immunoblot analysis of MCL1 protein expression in HMCLs

(n=3) overexpressing MCL1. HMCLs expressing empty vector were used as controls. -

actin was used as loading control. (E) MCL1 overexpression rescues AKT inhibitor-

induced cell death. HMCLs overexpressing MCL1 (n=3) were cultured for 3 days with

various concentrations of MK2206 (1.6; 3.2; 6.4 µM). HMCLs transduced with empty

vector were used as controls. Specific cell death was assessed by 7-AAD viability dye

staining and flow-cytometry. Means ± SEM of 3 independent experiments are shown

(****p<0.0001; one-way ANOVA with Bonferroni’s multiple comparison test). (F) Inhibition

of AKT sensitizes HMCLs (n=5) to MCL1 inhibitor induced cell death. Cells were treated


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for 3 days with various concentrations of MK2206 and S63845 (MCL1 inh.), as indicated

on the x-axis of the graphs. Percentages of specific cell death are depicted. Means ±

SEM of 3 experiments are shown. Chou-Talalay combination index (CI) values at ED75

(effective dose causing 75% cell death) are indicated in the graphs. (G) Inhibition of AKT

potentiates for MCL1 inhibitor-induced cell death in primary patient plasma cells (n=4).

Cells were treated for 3 days with a concentration of MK2206 and S63845 (2,5 µM

MK2206, 100 nM S63845 for AMC_4389, 100nM MK2206, 4nM S63845 for AMC_1345

and AMC_6615, 500 nM MK2206, 20nM S63845 for AMC_0713) either as single drug or

a combination. Means ± SEM of three technical replicates are shown. The Δ Bliss score

was calculated by subtracting the predicted cell death (Bliss) from the actual observed

effect of the combined inhibitors, -1 indicates an antagonistic effect and +1 indicates a

synergistic effect.


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0

20

40

60

80

100

[GSK2110183]%

spe

cific

cel

l dea

th

0.1 µM

0.2 µM

0.4 µM

0.8 µM

1.6 µM

3.2 µM

6.4µM

XG-3LME-1

LP-1MM1.S

XG-1OPM-2ANBL-6UM-3RPMI-8226

A

C

FIGURE 1

D

p-FOXO3(T32)p-FOXO1(T24)

FOXO1

AKT

p-GSKβ(S9)

p-AKT(S473)

β-Actin

MK2206

p-mTOR(S2448)

mTOR

S6

GSK3αGSK3β

p-AKT(T308)

FOXO3

p-S6

AMC0713 - +

AMC4389 - +

AMC1345

- +

AMC6615 - +

100 kDa80 kDa

80 kDa

100 kDa

60 kDa

60 kDa

46 kDa

45 kDa

289 kDa

289 kDa

60 kDa

46 kDa51 kDa

32 kDa

32 kDa

0

20

40

60

80

100%

spe

cific

cel

l dea

th

[MK2206]0.1

µM

0.2 µM

0.4 µM

0.8 µM

1.6 µM

3.2 µM

6.4µM

% s

peci

fic c

ell d

eath

AMC_438

9

AMC_186

4

AMC_994

6

AMC_071

3

AMC_134

5

AMC_661

50

20

40

60

80

100

****

**

****

***********

B

p-FOXO3(T32)

p-FOXO1(T24)

FOXO1

AKT

p-GSKβ(S9)

LME-1 MM1.S

p-AKT(S473)

β-Actin

MK2206p-mTOR(S2448)

mTOR

S6

GSK3αGSK3β

p-AKT(T308)

FOXO3

p-S6

- + - +XG-1 ANBL-6 - + - +

XG-3 LP-1 OPM-2 - + - + - +

UM-3 - +

RPMI-8226 - +

289 kDa

100 kDa80 kDa

80 kDa

100 kDa

60 kDa

60 kDa

46 kDa

45 kDa

289 kDa

60 kDa

46 kDa51 kDa

32 kDa

32 kDa


https://doi.org/10.1101/816694

CCS9 CCS9CCS9FOXO1

CCS9FOXO3

FOXO3

FOXO1

β-Actin

XG-3

LME-1

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 ****XG-1

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

**

XG-3

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 ***ANBL-6

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

**

MM1.S

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 * MK2206MK + FOXO1 inh.

A

B

C

DAMC4389

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

ns

AMC9946

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

****

AMC0713

% s

peci

fic c

ell d

eath

0

20

40

60

80

100****

MK2206MK + FOXO1 inh.

CCS9 CCS9CCS9FOXO1

CCS9CCS9FOXO3

LME-1

FOXO3

FOXO1

β-Actin

100 kDa

80 kDa

45 kDa

CCS9 CCS9FOXO1

CCS9FOXO3

FOXO3

FOXO1

β-Actin

MM1.S

FIGURE 2

AMC6615

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 ****

AMC1345

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 ***

LME-1

[MK2206]

% s

peci

fic c

ell d

eath

1.6 µM

0

20

40

60

80

100

****** ****

*****

ns

3.2 µM

6.4 µM

MM1.S

[MK2206]

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

ns**** ns

**** ns****

1.6 µM

3.2 µM

6.4 µM

XG-3

[MK2206]

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 ns**** ns

**** ns****

1.6 µM

3.2 µM

6.4 µM

Control

FOXO1 KO

FOXO3 KO


https://doi.org/10.1101/816694

ES= 0.45 NES= 1.38 Nominal P= 0.05FDR= 0.17

A

C

FIGURE 3

D

High FOXO activity

Thu Sep 26 13:01:16 2019 R2

Score-3 0 3

Tumor Myeloma - Hanamura - 542

MAS5.0 - u133p2

Low FOXO activity

B

Fri Oct 4 16:17:00 2019 R2

default1q21amp

alivenormal_contam

kmeans_kmeans

kmeans_roundsround_0round_1round_2round_3round_4round_5round_6round_7round_8round_9

gsm51284

gsm102632

gsm102633

gsm95804

gsm95730

gsm51298

gsm51336

gsm95697

gsm51074

gsm51165

gsm51034

gsm102630

gsm95767

gsm50991

gsm51322

gsm51285

gsm95662

gsm95657

gsm95769

gsm51122

gsm102610

gsm50986

gsm51247

gsm51036

gsm51053

gsm50989

gsm51198

gsm51108

gsm51232

gsm51172

gsm51035

gsm102629

gsm51027

gsm51070

gsm51250

gsm51328

gsm102634

gsm95818

gsm95737

gsm95802

gsm95708

gsm95740

gsm95743

gsm95819

gsm95817

gsm95815

gsm95696

gsm95768

gsm95728

gsm95744

gsm51248

gsm95764

gsm51315

gsm51327

gsm51098

gsm51217

gsm95791

gsm95741

gsm95746

gsm102624

gsm95794

gsm102627

gsm102616

gsm95807

gsm95795

gsm95796

gsm95777

gsm95809

gsm51325

gsm95778

gsm95653

gsm50995

gsm95664

gsm95748

gsm95766

gsm51245

gsm51281

gsm51004

gsm95782

gsm102628

gsm95759

gsm95733

gsm95698

gsm51230

gsm51049

gsm51064

gsm51319

gsm51264

gsm95805

gsm51302

gsm51069

gsm51138

gsm51231

gsm51150

gsm95688

gsm95772

gsm51161

gsm51177

gsm51139

gsm51073

gsm50998

gsm51081

gsm51179

gsm51128

gsm51193

gsm51043

gsm51185

gsm51114

gsm51194

gsm51059

gsm51182

gsm95707

gsm51042

gsm51063

gsm51218

gsm51029

gsm51204

gsm51192

gsm51115

gsm51078

gsm51012

gsm51009

gsm95810

gsm51005

gsm50996

gsm50993

gsm95646

gsm51019

gsm51289

gsm51318

gsm95719

gsm95718

gsm51240

gsm51208

gsm51263

gsm51234

gsm51295

gsm95797

gsm95798

gsm95770

gsm95773

gsm51294

gsm95754

gsm51089

gsm51087

gsm51120

gsm95816

gsm51037

gsm51071

gsm51033

gsm51266

gsm95725

gsm95669

gsm51058

gsm51329

gsm51119

gsm51267

gsm51028

gsm95701

gsm51220

gsm51304

gsm95685

gsm95745

gsm51183

gsm95774

gsm51111

gsm51317

gsm51056

gsm51052

gsm51116

gsm51077

gsm51061

gsm51025

gsm51176

gsm51181

gsm51148

gsm95706

gsm51088

gsm51016

gsm51309

gsm51262

gsm51144

gsm51277

gsm51072

gsm102620

gsm95753

gsm95647

gsm51038

gsm102617

gsm95732

gsm95679

gsm51261

gsm51255

gsm95709

gsm95760

gsm51021

gsm51200

gsm51196

gsm51299

gsm51062

gsm51018

gsm95824

gsm95822

gsm95821

gsm51333

gsm95687

gsm51207

gsm95813

gsm95739

gsm95721

gsm95814

gsm95806

gsm95735

gsm95665

gsm95736

gsm51323

gsm95765

gsm95826

gsm51286

gsm95785

gsm51060

gsm51047

gsm51044

gsm95812

gsm102621

gsm51092

gsm51206

gsm51100

gsm51093

gsm51030

gsm51162

gsm95652

gsm51305

gsm51212

gsm51118

gsm51215

gsm51110

gsm51157

gsm95704

gsm51147

gsm51014

gsm51252

gsm51095

gsm51256

gsm51127

gsm51013

gsm51296

gsm95790

gsm51090

gsm51195

gsm51189

gsm51175

gsm51159

gsm51155

gsm51149

gsm95755

gsm95710

gsm51068

gsm102612

gsm51011

gsm95673

gsm95703

gsm102625

gsm51094

gsm95776

gsm95695

gsm51332

gsm51241

gsm50990

gsm51107

gsm51000

gsm51167

gsm51133

gsm95654

gsm51103

gsm95683

gsm95792

gsm95716

gsm95788

gsm95779

gsm95692

gsm95650

gsm95787

gsm51040

gsm51334

gsm95731

gsm51131

gsm51109

gsm95761

gsm51168

gsm51236

gsm51055

gsm51050

gsm51276

gsm95751

gsm95738

gsm51243

gsm95752

gsm95648

gsm51265

gsm51271

gsm95786

gsm51024

gsm51301

gsm95801

gsm95775

gsm51274

gsm51253

gsm95781

gsm51282

gsm51275

gsm95649

gsm51242

gsm51283

gsm51141

gsm51257

gsm51310

gsm51003

gsm51268

gsm95723

gsm102623

gsm51320

gsm51102

gsm51101

gsm95820

gsm51097

gsm51143

gsm51221

gsm51158

gsm102613

gsm95690

gsm95678

gsm51229

gsm51105

gsm51084

gsm95780

gsm51260

gsm51235

gsm95823

gsm51321

gsm51066

gsm51222

gsm102618

gsm95680

gsm51278

gsm102611

gsm51239

gsm95783

gsm95691

gsm95727

gsm95675

gsm51022

gsm95793

gsm95729

gsm51051

gsm51225

gsm51316

gsm51065

gsm102607

gsm51288

gsm51171

gsm51330

gsm51306

gsm95684

gsm51297

gsm95722

gsm51209

gsm95784

gsm95651

gsm51174

gsm51154

gsm95693

gsm51326

gsm95724

gsm51184

gsm51140

gsm51311

gsm51205

gsm51113

gsm50988

gsm51312

gsm51287

gsm95661

gsm95659

gsm51290

gsm95717

gsm102606

gsm51314

gsm51 191

gsm102615

gsm51106

gsm51085

gsm51112

gsm95763

gsm51145

gsm51303

gsm51086

gsm51164

gsm51313

gsm95799

gsm95676

gsm51246

gsm95742

gsm95667

gsm95670

gsm51117

gsm51228

gsm51199

gsm95714

gsm51045

gsm51219

gsm95747

gsm95699

gsm95681

gsm51006

gsm51331

gsm51214

gsm95726

gsm51099

gsm51291

gsm95672

gsm51126

gsm51307

gsm95682

gsm51254

gsm51270

gsm95689

gsm51083

gsm51041

gsm51067

gsm95702

gsm51237

gsm50994

gsm50992

gsm51129

gsm51104

gsm51039

gsm51160

gsm51132

gsm51125

gsm51026

gsm51075

gsm50987

gsm95789

gsm95803

gsm51279

gsm51142

gsm51308

gsm51273

gsm51223

gsm51190

gsm95757

gsm95671

gsm95734

gsm95694

gsm95668

gsm102626

gsm51259

gsm51015

gsm95758

gsm95713

gsm51134

gsm51213

gsm51163

gsm51121

gsm51153

gsm95656

gsm51216

gsm51046

gsm51137

gsm51251

gsm51224

gsm51054

gsm51048

gsm51178

gsm51173

gsm95660

gsm51136

gsm51020

gsm50999

gsm50997

gsm51091

gsm51076

gsm51197

gsm51057

gsm51201

gsm51023

gsm51166

gsm51188

gsm95800

gsm51146

gsm51079

gsm51008

gsm51031

gsm51001

gsm51249

gsm95771

gsm51244

gsm51280

gsm51324

gsm51238

gsm51269

gsm51211

gsm51202

gsm51170

gsm95720

gsm95715

gsm51082

gsm51292

gsm51135

gsm51210

gsm51032

gsm51186

gsm51180

gsm102631

gsm51258

gsm51203

gsm51187

gsm51233

gsm51151

gsm95825

gsm51130

gsm51124

gsm51123

gsm51096

gsm51010

gsm95674

gsm95677

gsm51007

gsm95663

gsm95658

gsm51272

gsm95655

gsm102622

gsm51293

gsm95808

gsm95762

gsm95811

gsm51002

gsm95749

gsm102614

gsm102609

ALDOCPXN-AS1

PAGR1DUSP7

ADMENO2CMC2

FXNC11ORF24

PHPT1MRPL12

GPIPKM

TRIM14TAP2

PDE12MREG

NUP160LCMT2HAUS5PAQR4SSX2IPVMA21

SLC9A6STK26HPRT1PBDC1

C12ORF75XPO1

BARD1ACOT7POLQ

RAD51ATAD2TYMS

MYBL2HN1

APOOACLY

RRP15PAICS

KIF21ABTG3

SUMO3

Tumor Myeloma - Hanamura - 542MAS5.0 - u133p2

High FOXO actvity (n=387)Low FOXO activity (n=155)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

0 12 24 36 48 60

over

all s

urvi

val p

roba

bilit

y

Follow up in months

p=2.8e-05

MM cell lines GEPFOXO-dependent downregulation

360

1184 317

133 35

275

44

LME-1

MM1.S XG-3

ACLY ACOT7 ADM ALDOC APOO ATAD2 BARD1 BTG3 C11orf24 C12orf75 CMC2DUSP7 ENO2 FXN GPI HAUS5 HN1 HPRT1 KIF21A LCMT2 MREG MRPL12

MYBL2 NUP160 PAGR1 PAICS PAQR4 PBDC1 PDE12 PHPT1 PKM POLQ PXN-AS1RAD51 RRP15 SLC9A6 SSX2IP STK26 SUMO3 TAP2 TRIM14 TYMS VMA21 XPO1

MM cell lines GEPFOXO-dependent upregulation

457

562 328

60 32

145

23

LME-1

MM1.S XG-3

AGAP1 APPL2 ASB7 CITED2 CLK4 EPC2 ERBIN ERCC5 FAM193B GPBP1 HIST1H4H MANEA

MAP3K1 MPHOSPH8 NMRK1 PAN3 PLEKHA8 RAB2B RARRES3 SLC12A6 SLC33A1 SLC44A1 STK38


https://doi.org/10.1101/816694

ES= -0.54 NES= -1.71 Nominal P <0.001 FDR= 0.23

ES= -0.78 NES= -1.45 Nominal P= 0.03 FDR= 0.23

ES= -0.54 NES= -1.63 Nominal P= 0.01FDR= 0.05

A

B

C

DLME1 MM1.S XG-3

CDK4

C-MYC

Cyclin D2

β-Actin

Control FOXO1 KO Control FOXO3 KO Control FOXO3 KO

0/0.5/2.5 µMMK2206

30 kDa

60 kDa

45 kDa

30 kDa

LME-1

% c

ells

in p

hase

Control

Control +

MK

FOXO1 KO

FOXO1 KO +

MK0

20

40

60

80

100 *** nsnsMM1.S

Control

Control +

MK

FOXO3 KO

FOXO3 KO +

MK0

20

40

60

80

100 *** nsnsXG-3

Control

Control +

MK

FOXO3 KO

FOXO3 KO +

MK0

20

40

60

80

100

G1G2S**** ***ns

FIGURE 4

ANBL-6LME-1

LP-1

MM1.S XG-1XG-3

MK22062.5 μM

+ ++ +++- -- - - -

RPMI-

8226UM-3

+ +- -

% c

ells

in p

hase

0

20

40

60

80

100**** ** *** **** ** ns

G1G2S

β-Actin 45 kDa


https://doi.org/10.1101/816694

β-Actin

LME-1

p-FOXO1(T24)

MM1.S

β-Actin

p-FOXO3(T32)

LME-1

% c

ells

in p

hase

untreate

d

MK2206

GSK3 inh.

MK + GSK3 i

nh.0

20

40

60

80

100 * * nsLP-1

% c

ells

in p

hase

untreate

d

MK2206

GSK3 inh.

MK + GSK3 i

nh.0

20

40

60

80

100 ** * ns

G1G2S

XG-3

% c

ells

in p

hase

untreate

d

MK2206

GSK3 inh.

MK + GSK3 i

nh.0

20

40

60

80

100 * ** *MM1.S

% c

ells

in p

hase

untreate

d

MK2206

GSK3 inh.

MK + GSK3 i

nh.0

20

40

60

80

100 ** ns **

C

A

B

D

LME-1%

spe

cific

cel

l dea

th

0

20

40

60

80

100 ****LP-1

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

****

XG-1

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

**

XG-3

% s

peci

fic c

ell d

eath

0

20

40

60

80

100**** MK2206

MK + GSK3 inh.

MM1.S

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

***

AMC_4389

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

***

AMC_1864

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

**

AMC_9946

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

****

AMC_0713

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 ****AMC_6615

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 **** MK2206 MK + GSK3 inh.

80 kDa

45 kDa

100 kDa

45 kDa

FIGURE 5

XG-1

% c

ells

in p

hase

untreate

d

MK2206

GSK3 inh.

MK + GSK3 i

nh.0

20

40

60

80

100 ns ns ns

+GSK3 inh. no GSK3 inh. 0/0.5/2.5 µM MK2206

+GSK3 inh. no GSK3 inh. 0/0.5/2.5 µM MK2206


https://doi.org/10.1101/816694

LME-1

[MK2206/S63845 (nM)]

% s

peci

fic c

ell d

eath

0

50

100

100/3

.9

200/7

.81

400/1

5.62

800/3

1.25

1600

/62.5

3200

/125

6400

/250

MM1.S

[MK2206/S63845 (nM)]

% s

peci

fic c

ell d

eath

0

50

100

100/3

.90

200/7

.80

300/1

1.72

400/1

5.62

600/2

3.44

800/3

1.25

1200

/46.88

XG-3

[MK2206/S63845 (nM)]

% s

peci

fic c

ell d

eath

0

50

100

25/0.

97

50/1.

95

100/3

.90

200/7

.81

300/1

1.72

400/1

5.62

600/2

3.49

MK2206MCL1 inh.MK + MCL1 inh.

LME-1

MC

L1

EV MC

L1

EV MC

L1

EV

MM1.S XG-3

ED75CI: 0.23

ED75CI: 0.31

ED75CI: 0.43

F

ED

45 kDa

40 kDa

FIGURE 6

[MK2206]

% s

peci

fic c

ell d

eath

1.6 µM

3.2 µM

6.4 µM

0

20

40

60

80

100

LME-1

********

****

[MK2206]

% s

peci

fic c

ell d

eath

1.6 µM

3.2 µM

6.4 µM

0

20

40

60

80

100

MM1.S

********

****

[MK2206]

% s

peci

fic c

ell d

eath

1.6 µM

3.2 µM

6.4 µM

0

20

40

60

80

100

EVMCL1

XG-3

**** **** ****

MCL1

β-Actin

CHX CHX + MK22060 1 2 3 4 0 1 2 3 4

MM1.SCHX CHX + MK2206

0 1 2 3 4 0 1 2 3 4

XG-3

CHX CHX + MK22060 1 2 3 4 0 1 2 3 4 Hours

CHX CHX + MK22060 1 2 3 4 0 1 2 3 4 Hours

LME-1

UM-3CHX CHX + MK2206

0 1 2 3 4 0 1 2 3 4

RPMI-8226

10.0

30.0

10.0

00.1

6 10.0

30.0

00.0

00.2

4 10.3

50.1

80.0

50.6

8 10.3

40.1

20.0

30.4

2

10.3

10.0

50.0

50.5

7 10.0

60.0

00.0

00.4

5 10.7

00.4

60.3

10.8

8 10.1

60.0

80.0

20.6

8 10.3

80.1

30.0

00.7

0 10.0

00.0

00.0

00.0

0

45 kDa

40 kDa

45 kDa

40 kDa

XG-3

10.3

10.1

2 11.1

40.9

4 10.9

70.7

6

GSK3 inh. FOXO3 KOMM1.S

10.4

90.2

8 11.0

71.1

3 11.0

40.8

7

GSK3 inh. FOXO3 KOLME-1

10.8

30.6

1 11.1

31.2

6 10.9

60.9

7

GSK3 inh. FOXO1 KO

A0/0.5/2.5 µMMK2206

45 kDa

40 kDa MCL1

β-Actin β-ActinMCL1

MK2206- + - + - + - + - +

AMC4389

AMC1864

AMC9946

AMC0713

AMC1345

10.6

3 10.8

2 10.6

8 10.4

9 10.7

4

B

MCL1

β-Actin

MCL1

β-Actin

C

13.7

8 12.0

8 110

.33

GAMC4389

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

AMC1345

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

AMC6615

% s

peci

fic c

ell d

eath

0

20

40

60

80

100

AMC0713

% s

peci

fic c

ell d

eath

0

20

40

60

80

100 MK2206MCL1 inh.MK + MCL1 inh.

-1

Δ Bliss scoreAMC_4389 0,453AMC_1345 0,016AMC_6615 0,024AMC_0713 0,170

1

[MK2206/S63845 (nM)]10

0/3.9

200/7

.81

400/1

5.62

800/3

1.25

1600

/62.5

3200

/125

6400

/250

% s

peci

fic c

ell d

eath

0

50

100UM-3

[MK2206/S63845 (nM)]10

0/3.9

200/7

.81

400/1

5.62

800/3

1.25

1600

/62.5

3200

/125

6400

/250

% s

peci

fic c

ell d

eath

0

50

100RPMI-8226

ED75CI: 0.51

ED75CI: 0.48


https://doi.org/10.1101/816694

targeting akt elicits tumor suppressive functions of foxo ...multiple myeloma (mm) is a malignancy...

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