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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: j.e.guikema@amsterdamumc.nl, phone: +31-20-5665708, fax: +31-
20-5669523
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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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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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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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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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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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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
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
31
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.
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
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
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
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
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
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
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
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
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
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
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
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
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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