bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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
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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.
18 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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.
19 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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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.
24 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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.
25 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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.
26 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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
27 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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
28 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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
29 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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
30 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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.
31 FIGURE 1 A B 100 100 XG-3 100 **** bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which*** was not LME-1 **** 80 certified by peer review)80 is the author/funder. All rights reserved. No reuse allowed without permission. MM1.S 80 LP-1 60 60 60 XG-1 40 40 OPM-2 40 **** ANBL-6 ** 20 20 UM-3 20 % specific cell death % specific cell death % specific cell death **** 0 0 RPMI-8226 0
0.1 µM 0.2 µM 0.4 µM 0.8 µM 1.6 µM 3.2 µM 6.4µM 0.1 µM 0.2 µM 0.4 µM 0.8 µM 1.6 µM 3.2 µM 6.4µM
[MK2206] [GSK2110183] AMC_4389AMC_1864AMC_9946AMC_0713AMC_1345AMC_6615 C LME-1 MM1.S XG-1 XG-3 ANBL-6 LP-1 OPM-2 UM-3 RPMI-8226 MK2206 - + - + - + - + - + - + - + - + - + p-mTOR(S2448) 289 kDa mTOR 289 kDa
p-FOXO3(T32) 100 kDa 80 kDa p-FOXO1(T24) FOXO3 100 kDa 80 kDa FOXO1 p-AKT(S473) 60 kDa
p-AKT(T308) 60 kDa AKT 60 kDa
p-GSKβ(S9) 46 kDa GSK3α 51 kDa GSK3β 46 kDa p-S6 32 kDa S6 32 kDa β-Actin 45 kDa D AMC AMC AMC AMC 4389 0713 1345 6615 MK2206 - + - + - + - + p-mTOR(S2448) 289 kDa
mTOR 289 kDa
p-FOXO3(T32) 100 kDa p-FOXO1(T24) 80 kDa FOXO3 100 kDa
FOXO1 80 kDa
p-AKT(S473) 60 kDa
p-AKT(T308) 60 kDa
AKT 60 kDa
p-GSKβ(S9) 46 kDa GSK3α 51 kDa GSK3β 46 kDa p-S6 32 kDa
S6 32 kDa
β-Actin 45 kDa FIGURE 2 A LME-1 MM1.S XG-3 bioRxivCCS9 preprintCCS9 doi:CCS9 https://doi.org/10.1101/816694CCS9; this versionCCS9 posted OctoberCCS9 24, 2019. The copyrightCCS9 holderCCS9CCS9 for thisCCS9 preprint (which was not FOXO1FOXO3certified by peer review) is the author/funder.FOXO1 All rightsFOXO3 reserved. No reuse allowed withoutFOXO1 permission.FOXO3
100 kDa FOXO3 FOXO3 FOXO3
80 kDa FOXO1 FOXO1 FOXO1 45 kDa β-Actin β-Actin β-Actin B LME-1 MM1.S XG-3 **** **** **** **** ns 100 ns 100 ns 100 ns **** ns Control *** **** ns 80 ** 80 80 FOXO1 KO ** ns **** FOXO3 KO **** 60 60 60
40 40 40
20 20 20 % specific cell death % specific cell death % specific cell death 0 0 0
1.6 µM 3.2 µM 6.4 µM 1.6 µM 3.2 µM 6.4 µM 1.6 µM 3.2 µM 6.4 µM [MK2206] [MK2206] [MK2206] C LME-1 XG-3 ANBL-6 XG-1 MM1.S MK2206 100 100 *** 100 100 100 * **** MK + FOXO1 inh. 80 80 80 80 80 ** 60 60 60 60 60
40 40 40 40 40 ** 20 20 20 20 20 % specific cell death % specific cell death % specific cell death % specific cell death % specific cell death 0 0 0 0 0 D AMC AMC AMC AMC AMC 4389 9946 0713 1345 6615
100 100 100 100 *** 100 **** MK2206 **** MK + FOXO1 inh. 80 80 80 80 80
60 60 60 60 60 **** 40 40 40 40 40
20 ns 20 20 20 20 % specific cell death % specific cell death % specific cell death % specific cell death % specific cell death 0 0 0 0 0 FIGURE 3 A B MM cell lines GEP MM cell lines GEP ACLY MYBL2 FOXO-dependent upregulation FOXO-dependent downregulation ACOT7 NUP160 bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ADM PAGR1 ALDOC PAICS ES= 0.45 APOO PAQR4
NES= 1.38 ATAD2 PBDC1 Nominal P= 0.05 457 AGAP1 MAP3K1 360 BARD1 PDE12 LME-1 APPL2 MPHOSPH8 LME-1 BTG3 PHPT1 FDR= 0.17 ASB7 NMRK1 C11orf24 PKM CITED2 PAN3 133 C12orf75 POLQ CLK4 PLEKHA8 35 CMC2 PXN-AS1
60 44 DUSP7 RAD51 23 32 EPC2 RAB2B ERBIN RARRES3 ENO2 RRP15 ERCC5 SLC12A6 FXN SLC9A6
FAM193B SLC33A1 MM1.S XG-3 GPI SSX2IP MM1.S145 XG-3 GPBP1 SLC44A1 1184 275 317 HAUS5 STK26 562 328 HIST1H4H STK38 HN1 SUMO3
MANEA HPRT1 TAP2 KIF21A TRIM14 LCMT2 TYMS MREG VMA21 MRPL12 XPO1
C
High FOXO activity Tumor Myeloma - Hanamura - 542 Low FOXO activity gsm102632 gsm102633 gsm102630 gsm102610 gsm102629 gsm102634 gsm102624 gsm102627 gsm102616 gsm102628 gsm102612 gsm102625 gsm102623 gsm102613 gsm102618 gsm102607 gsm102606 gsm102615 gsm102626 gsm102631 gsm102622 gsm102614 gsm102609 gsm102620 gsm102617 gsm102621 gsm102611 gsm51284 gsm95804 gsm95730 gsm51298 gsm51336 gsm95697 gsm51074 gsm51034 gsm95767 gsm50991 gsm51322 gsm51285 gsm95662 gsm95657 gsm95769 gsm50986 gsm51247 gsm51036 gsm51053 gsm50989 gsm51232 gsm51035 gsm51027 gsm51070 gsm51250 gsm51328 gsm95818 gsm95737 gsm95802 gsm95708 gsm95740 gsm95743 gsm95819 gsm95817 gsm95815 gsm95696 gsm95768 gsm95728 gsm95744 gsm51248 gsm95764 gsm51315 gsm51327 gsm51098 gsm51217 gsm95791 gsm95741 gsm95746 gsm95794 gsm95807 gsm95795 gsm95796 gsm95777 gsm95809 gsm51325 gsm95778 gsm95653 gsm50995 gsm95664 gsm95748 gsm95766 gsm51245 gsm51281 gsm51004 gsm95782 gsm95759 gsm95733 gsm95698 gsm51230 gsm51049 gsm51064 gsm51319 gsm51264 gsm95805 gsm51302 gsm51069 gsm51231 gsm95688 gsm95772 gsm51073 gsm50998 gsm51081 gsm51043 gsm51059 gsm95707 gsm51042 gsm51063 gsm51218 gsm51029 gsm51204 gsm95704 gsm51014 gsm51252 gsm51095 gsm51256 gsm51013 gsm51296 gsm95790 gsm51090 gsm95755 gsm95710 gsm51068 gsm95673 gsm95703 gsm51094 gsm95776 gsm95695 gsm51332 gsm51241 gsm50990 gsm51000 gsm95654 gsm95683 gsm95792 gsm95716 gsm95788 gsm95779 gsm95692 gsm95650 gsm95787 gsm51040 gsm51334 gsm95731 gsm95761 gsm51236 gsm51055 gsm51050 gsm51276 gsm95751 gsm95738 gsm51243 gsm95752 gsm95648 gsm51265 gsm51271 gsm95786 gsm51024 gsm51301 gsm95801 gsm95775 gsm51274 gsm51253 gsm95781 gsm51282 gsm51275 gsm95649 gsm51242 gsm51283 gsm51257 gsm51310 gsm51003 gsm51268 gsm95723 gsm51320 gsm95820 gsm51097 gsm51221 gsm95690 gsm95678 gsm51229 gsm51084 gsm95780 gsm51260 gsm51235 gsm95823 gsm51321 gsm51066 gsm51222 gsm95680 gsm51278 gsm51239 gsm95783 gsm95691 gsm95727 gsm95675 gsm51022 gsm95793 gsm95729 gsm51051 gsm51225 gsm51316 gsm51065 gsm51288 gsm51330 gsm51306 gsm95684 gsm51297 gsm95722 gsm51209 gsm95784 gsm95651 gsm95693 gsm51326 gsm95724 gsm51205 gsm50988 gsm51312 gsm51287 gsm95661 gsm95659 gsm51290 gsm95717 gsm51314 gsm51085 gsm95763 gsm51303 gsm51086 gsm51313 gsm95799 gsm95676 gsm51246 gsm95742 gsm95667 gsm95670 gsm51228 gsm95714 gsm51045 gsm51219 gsm95747 gsm95699 gsm95681 gsm51006 gsm51331 gsm51214 gsm95726 gsm51099 gsm51291 gsm95672 gsm51307 gsm95682 gsm51254 gsm51270 gsm95689 gsm51083 gsm51041 gsm51067 gsm95702 gsm51237 gsm50994 gsm50992 gsm51039 gsm51026 gsm51075 gsm50987 gsm95789 gsm95803 gsm51279 gsm51308 gsm51273 gsm51223 gsm95757 gsm95671 gsm95734 gsm95694 gsm95668 gsm51259 gsm51015 gsm95758 gsm95713 gsm51213 gsm95656 gsm51216 gsm51046 gsm51251 gsm51224 gsm51054 gsm51048 gsm95660 gsm51020 gsm50999 gsm50997 gsm51091 gsm51076 gsm51057 gsm51201 gsm51023 gsm95800 gsm51079 gsm51008 gsm51031 gsm51001 gsm51249 gsm95771 gsm51244 gsm51280 gsm51324 gsm51238 gsm51269 gsm51202 gsm95720 gsm95715 gsm51082 gsm51292 gsm51210 gsm51032 gsm51258 gsm51203 gsm51233 gsm95825 gsm51096 gsm51010 gsm95674 gsm95677 gsm51007 gsm95663 gsm95658 gsm51272 gsm95655 gsm51293 gsm95808 gsm95762 gsm51002 gsm95749 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 gsm95816 gsm51037 gsm51071 gsm51033 gsm51266 gsm95725 gsm95669 gsm51058 gsm51329 gsm51267 gsm51028 gsm95701 gsm51220 gsm51304 gsm95685 gsm95745 gsm95774 gsm51317 gsm51056 gsm51052 gsm51077 gsm51061 gsm51025 gsm95706 gsm51088 gsm51016 gsm51309 gsm51262 gsm51277 gsm51072 gsm95753 gsm95647 gsm51038 gsm95732 gsm95679 gsm51261 gsm51255 gsm95709 gsm95760 gsm51021 gsm51200 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 gsm51092 gsm51206 gsm51093 gsm51030 gsm95652 gsm51305 gsm51212 gsm51215 gsm51165 gsm51122 gsm51198 gsm51108 gsm51172 gsm51138 gsm51150 gsm51161 gsm51177 gsm51139 gsm51179 gsm51128 gsm51193 gsm51185 gsm51194 gsm51182 gsm51192 gsm51157 gsm51147 gsm51127 gsm51195 gsm51189 gsm51175 gsm51159 gsm51155 gsm51149 gsm51011 gsm51107 gsm51167 gsm51133 gsm51103 gsm51131 gsm51109 gsm51168 gsm51141 gsm51102 gsm51101 gsm51143 gsm51158 gsm51105 gsm51171 gsm51174 gsm51154 gsm51184 gsm51140 gsm51311 gsm51 gsm51106 gsm51145 gsm51164 gsm51199 gsm51126 gsm51129 gsm51104 gsm51160 gsm51132 gsm51125 gsm51142 gsm51190 gsm51134 gsm51163 gsm51121 gsm51153 gsm51137 gsm51178 gsm51173 gsm51136 gsm51197 gsm51166 gsm51188 gsm51146 gsm51211 gsm51170 gsm51135 gsm51186 gsm51180 gsm51187 gsm51151 gsm51130 gsm51124 gsm51123 gsm95811 gsm51120 gsm51183 gsm51176 gsm51181 gsm51148 gsm51144 gsm51196 gsm51100 gsm51162 gsm51113 gsm51112 gsm51117 gsm51114 gsm51115 gsm51119 gsm51116 gsm51118 gsm51110 gsm51111 191
default 1q21amp alive normal_contam kmeans _kmeans kmeans_rounds round_0 round_1 round_2 round_3 round_4 round_5 round_6 round_7 round_8 round_9 ALDOC PXN-AS1 PAGR1 DUSP7 ADM ENO2 CMC2 FXN C11ORF24 PHPT1 MRPL12 GPI MAS5.0 - u133p2 PKM TRIM14 TAP2 PDE12 MREG NUP160 LCMT2 HAUS5 PAQR4 SSX2IP VMA21 SLC9A6 STK26 HPRT1 PBDC1 C12ORF75 XPO1 BARD1 ACOT7 POLQ RAD51 ATAD2 TYMS MYBL2 HN1 Thu Sep 26 13:01:16 2019 R2 APOO ACLY RRP15 PAICS KIF21A BTG3 SUMO3
-3 0 3 Score Fri Oct 4 16:17:00 2019 R2
D Tumor Myeloma - Hanamura - 542 MAS5.0 - u133p2
1.00 High FOXO actvity (n=387) Low FOXO activity (n=155) 0.90
0.80
0.70
0.60
0.50
0.40
0.30 overall survival probability
0.20
0.10 p=2.8e-05
0.00 0 12 24 36 48 60
Follow up in months FIGURE 4 A bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
ES= -0.54 ES= -0.78 ES= -0.54 NES= -1.71 NES= -1.45 NES= -1.63 Nominal P <0.001 Nominal P= 0.03 Nominal P= 0.01 FDR= 0.23 FDR= 0.23 FDR= 0.05
B ** *** ** *** ** * ** ns 100 S G2 80 G1
60
40
20
% cells in phase 0 -+ -- ++ - - - +++ --+ + MK2206 2.5 μM
XG-3 LP-1 XG-1 UM-3 RPMI- LME-1 MM1.S ANBL-6 8226 C LME-1 MM1.S XG-3 ns ns ns *** ns ns *** **** *** 100 100 100 S G2 80 80 80 G1 60 60 60
40 40 40
% cells in phase 20 20 20
0 0 0
Control Control Control FOXO1 KO FOXO3 KO FOXO3 KO Control + MK Control + MK Control + MK FOXO1 KO + MK FOXO3 KO + MK FOXO3 KO + MK D LME1 MM1.S XG-3 Control FOXO1 KO Control FOXO3 KO Control FOXO3 KO
0/0.5/2.5 µM MK2206
CDK4 30 kDa C-MYC 60 kDa
β-Actin 45 kDa
Cyclin D2 30 kDa
β-Actin 45 kDa FIGURE 5 A LME-1 XG-3 LP-1 XG-1 MM1.S bioRxiv preprint doi: https://doi.org/10.1101/816694; this version posted October 24, 2019. The copyright holder for this preprint (which was not 100 **** certified100 by peer review) is the100 author/funder. All rights 100reserved. No reuse allowed100 without permission. MK2206 **** MK + GSK3 inh. 80 80 80 80 80
60 60 60 60 60 *** 40 40 40 **** 40 ** 40 20 20 20 20 20 % specific cell death % specific cell death % specific cell death % specific cell death % specific cell death 0 0 0 0 0
B AMC_4389 AMC_1864 AMC_9946 AMC_0713 AMC_6615 **** 100 100 100 100 **** 100 MK2206 MK + GSK3 inh. 80 80 80 80 80
60 60 60 60 60 **** 40 40 40 40 40 ** 20 20 20 20 20 % specific cell death % specific cell death *** % specific cell death % specific cell death % specific cell death 0 0 0 0 0 C LME-1 LP-1 XG-1 ns ns ns ns ns 100 * * 100 ** * 100
80 80 80
60 60 60
40 40 40
20 20 20 % cells in phase % cells in phase 0 % cells in phase 0 0
MK2206 MK2206 MK2206 untreated GSK3 inh. untreated GSK3 inh. untreated GSK3 inh.
MK + GSK3 inh. MK + GSK3 inh. MK + GSK3 inh.
XG-3 MM1.S * ** * ** ns ** 100 100 S 80 80 G2 G1 60 60
40 40
20 20
% cells in phase 0 % cells in phase 0
MK2206 MK2206 untreated GSK3 inh. untreated GSK3 inh.
MK + GSK3 inh. MK + GSK3 inh. D LME-1 MM1.S no GSK3 inh. +GSK3 inh. no GSK3 inh. +GSK3 inh. 0/0.5/2.5 µM 0/0.5/2.5 µM MK2206 MK2206
80 kDa p- FOXO1(T24) 100 kDa p-FOXO3(T32)
45 kDa β- Actin 45 kDa β-Actin FIGURE 6 A B LME-1 MM1.S XG-3 AMC AMC AMC AMC AMC bioRxiv preprintGSK3 doi: inh. https://doi.org/10.1101/816694FOXO1 KO GSK3 inh. FOXO3; this KO version postedGSK3 October inh. FOXO3 24, KO 2019. The copyright4389 1864 holder9946 for this0713 preprint1345 (which was not certified by peer review) is the author/funder. All rights reserved. No0/0.5/2.5 reuse µM allowed without permission. - + - + - + - + - + MK2206 MK2206 40 kDa MCL1 MCL1 45 kDa β-Actin β-Actin
1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.83 0.61 1.13 1.26 0.96 0.97 0.49 0.28 1.07 1.13 1.04 0.87 0.31 0.12 1.140.94 0.97 0.76 0.63 0.82 0.68 0.49 0.74 C LME-1 MM1.S XG-3 CHX CHX + MK2206 CHX CHX + MK2206 CHX CHX + MK2206 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Hours 40 kDa MCL1
45 kDa β-Actin
1 1 1 1 1 1 0.57 0.31 0.05 0.05 0.45 0.06 0.00 0.00 0.88 0.70 0.46 0.31 0.68 0.16 0.08 0.02 0.70 0.38 0.13 0.00 0.00 0.00 0.00 0.00
UM-3 RPMI-8226 CHX CHX + MK2206 CHX CHX + MK2206 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Hours 40 kDa MCL1
45 kDa β-Actin
1 1 1 1 0.16 0.03 0.01 0.00 0.24 0.03 0.00 0.00 0.68 0.35 0.18 0.05 0.42 0.34 0.12 0.03 D E LME-1 MM1.S XG-3 LME-1 MM1.S XG-3 100 **** 100 100 **** **** **** EV MCL1 EV MCL1 EV MCL1 80 **** 80 **** 80 EV MCL1 40 kDa MCL1 **** 60 60 **** 60 45 kDa β-Actin 40 **** 40 40
1 1 1 3.78 2.08 20 20 20 10.33 % specific cell death % specific cell death % specific cell death 0 0 0
1.6 µM 3.2 µM 6.4 µM 1.6 µM 3.2 µM 6.4 µM 1.6 µM 3.2 µM 6.4 µM [MK2206] [MK2206] [MK2206] F LME-1 MM1.S XG-3 100 ED75 100 ED75 100 ED75 CI: 0.23 CI: 0.31 CI: 0.43
50 50 50 % specific cell death % specific cell death % specific cell death 0 0 0
100/3.9200/7.81 25/0.97 50/1.95 400/15.62800/31.251600/62.53200/1256400/250 100/3.90 200/7.80 100/3.90 200/7.81300/11.72400/15.62600/23.49 300/11.72400/15.62600/23.44800/31.251200/46.88 [MK2206/S63845 (nM)] [MK2206/S63845 (nM)] [MK2206/S63845 (nM)] UM-3 RPMI-8226 100 100 ED75 ED75 MK2206 CI: 0.51 CI: 0.48 MCL1 inh. MK + MCL1 inh.
50 50 % specific cell death % specific cell death 0 0
100/3.9 100/3.9 200/7.81400/15.62800/31.251600/62.53200/1256400/250 200/7.81400/15.62800/31.251600/62.53200/1256400/250 [MK2206/S63845 (nM)] [MK2206/S63845 (nM)] G AMC AMC AMC AMC 4389 1345 6615 0713 100 100 100 100 MK2206 Δ Bliss score MCL1 inh. 80 80 80 80 MK + MCL1 inh. AMC_4389 0,453 AMC_1345 0,016 60 60 60 60 AMC_6615 0,024 40 40 40 40 AMC_0713 0,170
20 20 20 20 -1 1 % specific cell death % specific cell death % specific cell death % specific cell death 0 0 0 0