Introduction: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. Methods: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. Results: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Conclusion: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

1.
Berliner
N
.
Lessons from congenital neutropenia: 50 years of progress in understanding myelopoiesis
.
Blood
.
2008
;
111
(
12
):
5427
32
.
2.
Gutierrez
MG
,
Master
SS
,
Singh
SB
,
Taylor
GA
,
Colombo
MI
,
Deretic
V
.
Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages
.
Cell
.
2004
;
119
(
6
):
753
66
.
3.
Sanjuan
MA
,
Dillon
CP
,
Tait
SWG
,
Moshiach
S
,
Dorsey
F
,
Connell
S
, et al
.
Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis
.
Nature
.
2007
;
450
(
7173
):
1253
7
.
4.
Jacquel
A
,
Obba
S
,
Solary
E
,
Auberger
P
.
Proper macrophagic differentiation requires both autophagy and caspase activation
.
Autophagy
.
2012
;
8
(
7
):
1141
3
.
5.
Zhang
C
,
Cuervo
AM
.
Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function
.
Nat Med
.
2008
;
14
(
9
):
959
65
.
6.
Deretic
V
,
Levine
B
.
Autophagy balances inflammation in innate immunity
.
Autophagy
.
2018
;
14
(
2
):
243
51
.
7.
Warr
MR
,
Binnewies
M
,
Flach
J
,
Reynaud
D
,
Garg
T
,
Malhotra
R
, et al
.
FOXO3A directs a protective autophagy program in haematopoietic stem cells
.
Nature
.
2013
;
494
(
7437
):
323
7
.
8.
Liu
J
,
Bi
G
,
Wen
P
,
Yang
W
,
Ren
X
,
Tang
T
, et al
.
Down-regulation of CD44 contributes to the differentiation of HL-60 cells induced by ATRA or HMBA
.
Cell Mol Immunol
.
2007
;
4
(
1
):
59
63
.
9.
Mortensen
M
,
Soilleux
EJ
,
Djordjevic
G
,
Tripp
R
,
Lutteropp
M
,
Sadighi-Akha
E
, et al
.
The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance
.
J Exp Med
.
2011
;
208
(
3
):
455
67
.
10.
Watson
A
,
Riffelmacher
T
,
Stranks
A
,
Williams
O
,
De Boer
J
,
Cain
K
, et al
.
Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia
.
Cell Death Discov
.
2015
;
1
(
1
):
15008
10
.
11.
Tanida
I
.
Autophagy basics
.
Microbiol Immunol
.
2011
;
55
(
1
):
1
11
.
12.
Bandyopadhyay
U
,
Kaushik
S
,
Varticovski
L
,
Cuervo
AM
.
The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane
.
Mol Cell Biol
.
2008
;
28
(
18
):
5747
63
.
13.
Kaushik
S
,
Cuervo
AM
.
The coming of age of chaperone-mediated autophagy
.
Nat Rev Mol Cell Biol
.
2018
;
19
(
6
):
365
81
.
14.
Cecconi
F
,
Levine
B
.
The role of autophagy in mammalian development: cell makeover rather than cell death
.
Dev Cell
.
2008
;
15
(
3
):
344
57
.
15.
Eskelinen
EL
.
New insights into the mechanisms of macroautophagy in mammalian cells
.
Int Rev Cell Mol Biol
.
2008
;
266
:
207
47
.
16.
Zeng
M
,
Zhou
JN
.
Roles of autophagy and mTOR signaling in neuronal differentiation of mouse neuroblastoma cells
.
Cell Signal
.
2008
;
20
(
4
):
659
65
.
17.
Salemi
S
,
Yousefi
S
,
Constantinescu
MA
,
Fey
MF
,
Simon
HU
.
Autophagy is required for self-renewal and differentiation of adult human stem cells
.
Cell Res
.
2012
;
22
(
2
):
432
5
.
18.
Qu
X
,
Yu
J
,
Bhagat
G
,
Furuya
N
,
Hibshoosh
H
,
Troxel
A
, et al
.
Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene
.
J Clin Invest
.
2003
;
112
(
12
):
1809
20
.
19.
Hoare
M
,
Young
ARJ
,
Narita
M
.
Autophagy in cancer: having your cake and eating it
.
Semin Cancer Biol
.
2011
;
21
(
6
):
397
404
.
20.
de The
H
,
Esnault
C
,
Geoffroy
MC
.
Retinoids in haematology: a timely revival
.
2021
.
21.
Zhu
J
,
Gianni
M
,
Kopf
E
,
Honoré
N
,
Chelbi-Alix
M
,
Koken
M
, et al
.
Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor α (RARα) and oncogenic RARα fusion proteins
.
Proc Natl Acad Sci USA
.
1999
;
96
(
26
):
14807
12
.
22.
Isakson
P
,
Bjørås
M
,
Bøe
SO
,
Simonsen
A
.
Autophagy contributes to therapy-induced degradation of the PML/RARA oncoprotein
.
Blood
.
2010
;
116
(
13
):
2324
31
.
23.
Anguiano
J
,
Garner
TP
,
Mahalingam
M
,
Das
BC
,
Gavathiotis
E
,
Cuervo
AM
.
Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives
.
Nat Chem Biol
.
2013
;
9
(
6
):
374
82
.
24.
Massey
AC
,
Kaushik
S
,
Cuervo
AM
.
Lysosomal chat maintains the balance
.
Autophagy
.
2006
;
2
(
4
):
325
7
.
25.
Kon
M
,
Kiffin
R
,
Koga
H
,
Chapochnick
J
,
Macian
F
,
Varticovski
L
, et al
.
Chaperone-mediated autophagy is required for tumor growth
.
Sci Transl Med
.
2011
;
3
(
109
):
109ra117
.
26.
Tasset
I
,
Cuervo
AM
.
Role of chaperone-mediated autophagy in metabolism
.
FEBS J
.
2016
;
283
(
13
):
2403
13
.
27.
Lv
L
,
Li
D
,
Zhao
D
,
Lin
R
,
Chu
Y
,
Zhang
H
, et al
.
Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth
.
Mol Cell
.
2011
;
42
(
6
):
719
30
.
28.
Xia
HG
,
Najafov
A
,
Geng
J
,
Galan-Acosta
L
,
Han
X
,
Guo
Y
, et al
.
Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death
.
J Cell Biol
.
2015
;
210
(
5
):
705
16
.
29.
Dong
S
,
Wang
Q
,
Kao
YR
,
Diaz
A
,
Tasset
I
,
Kaushik
S
, et al
.
Chaperone-mediated autophagy sustains haematopoietic stem-cell function
.
Nature
.
2021
;
591
(
7848
):
117
23
.
30.
Robert
G
,
Jacquel
A
,
Auberger
P
.
Chaperone-mediated autophagy and its emerging role in hematological malignancies
.
Cells
.
2019
;
8
(
10
):
1260
.
31.
Swan
C
,
Bühler
B
,
Steinberger
P
,
Tschan
MP
,
Barbas
CF
3rd
,
Torbett
BE
.
T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery
.
Gene Ther
.
2006
;
13
(
20
):
1480
92
.
32.
Brigger
D
,
Proikas-Cezanne
T
,
Tschan
MP
.
WIPI-Dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells
.
Cell Death Dis
.
2014
;
5
(
7
):
e1315
.
33.
Rizzi
M
,
Tschan
MP
,
Britschgi
C
,
Britschgi
A
,
Hügli
B
,
Grob
TJ
, et al
.
The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells
.
J Leukoc Biol
.
2007
;
81
(
6
):
1599
608
.
34.
Reinhart
R
,
Wicki
S
,
Kaufmann
T
.
In vitro differentiation of mouse granulocytes
.
Methods Mol Biol
.
2016
;
1419
:
95
107
.
35.
Ho
PWL
,
Leung
CT
,
Liu
H
,
Pang
SYY
,
Lam
CSC
,
Xian
J
, et al
.
Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA)
.
Autophagy
.
2020
;
16
(
2
):
347
70
.
36.
Bagger
FO
,
Sasivarevic
D
,
Sohi
SH
,
Laursen
LG
,
Pundhir
S
,
Sønderby
CK
, et al
.
BloodSpot: a database of gene expression profiles and transcriptional programs for healthy and malignant haematopoiesis
.
Nucleic Acids Res
.
2016
;
44
(
D1
):
D917
24
.
37.
Jin
J
,
Britschgi
A
,
Schläfli
AM
,
Humbert
M
,
Shan-Krauer
D
,
Batliner
J
, et al
.
Low autophagy (ATG) gene expression is associated with an immature AML blast cell phenotype and can Be restored during AML differentiation therapy
.
Oxid Med Cell Longev
.
2018
;
2018
:
1482795
.
38.
Patel
B
,
Cuervo
AM
.
Methods to study chaperone-mediated autophagy
.
Methods
.
2015
;
75
:
133
40
.
39.
Koga
H
,
Martinez-Vicente
M
,
Arias
E
,
Kaushik
S
,
Sulzer
D
,
Cuervo
AM
.
Constitutive upregulation of chaperone-mediated autophagy in Huntington’s disease
.
J Neurosci
.
2011
;
31
(
50
):
18492
505
.
40.
Humbert
M
,
Federzoni
EA
,
Tschan
MP
.
Distinct TP73–DAPK2–ATG5 pathway involvement in ATO-mediated cell death versus ATRA-mediated autophagy responses in APL
.
J Leukoc Biol
.
2017
;
102
(
6
):
1357
70
.
41.
Duprez
E
,
Lillehaug
JR
,
Naoe
T
,
Lanotte
M
.
cAMP signalling is decisive for recovery of nuclear bodies (PODs) during maturation of RA-resistant t(15;17) promyelocytic leukemia NB4 cells expressing PML-RAR alpha
.
Oncogene
.
1996
;
12
(
11
):
2451
9
.
42.
Pendino
F
,
Flexor
M
,
Delhommeau
F
,
Buet
D
,
Lanotte
M
,
Segal-Bendirdjian
E
.
Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation
.
Proc Natl Acad Sci USA
.
2001
;
98
(
12
):
6662
7
.
43.
Choi
HS
,
Kim
JW
,
Cha
YN
,
Kim
C
.
A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells
.
J Immunoassay Immunochem
.
2006
;
27
(
1
):
31
44
.
44.
Page
N
,
Gros
F
,
Schall
N
,
Décossas
M
,
Bagnard
D
,
Briand
JP
, et al
.
HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus
.
Ann Rheum Dis
.
2011
;
70
(
5
):
837
43
.
45.
Macri
C
,
Wang
F
,
Tasset
I
,
Schall
N
,
Page
N
,
Briand
JP
, et al
.
Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
.
Autophagy
.
2015
;
11
(
3
):
472
86
.
46.
Wang
F
,
Tasset
I
,
Cuervo
AM
,
Muller
S
.
In vivo remodeling of altered autophagy-lysosomal pathway by a phosphopeptide in lupus
.
Cells
.
2020
;
9
(
10
):
2328
15
.
47.
Riffelmacher
T
,
Clarke
A
,
Richter
FC
,
Stranks
A
,
Pandey
S
,
Danielli
S
, et al
.
Autophagy-dependent generation of free fatty acids is critical for normal neutrophil differentiation
.
Immunity
.
2017
;
47
(
3
):
466
80.e5
.
48.
Peng
J
,
Han
SM
,
Chen
ZH
,
Yang
J
,
Pei
YQ
,
Bao
C
, et al
.
Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells
.
Biochem Biophys Res Commun
.
2020
;
522
(
2
):
348
54
.
49.
Suzuki
J
,
Nakajima
W
,
Suzuki
H
,
Asano
Y
,
Tanaka
N
.
Chaperone-mediated autophagy promotes lung cancer cell survival through selective stabilization of the pro-survival protein, MCL1
.
Biochem Biophys Res Commun
.
2017
;
482
(
4
):
1334
40
.
50.
Arias
E
,
Koga
H
,
Diaz
A
,
Mocholi
E
,
Patel
B
,
Cuervo
AM
.
Lysosomal mTORC2/PHLPP1/akt regulate chaperone-mediated autophagy
.
Mol Cell
.
2015
;
59
(
2
):
270
84
.
51.
Koga
H
,
Martinez-Vicente
M
,
MacIan
F
,
Verkhusha
VV
,
Cuervo
AM
.
A photoconvertible fluorescent reporter to track chaperone-mediated autophagy
.
Nat Commun
.
2011
;
2
(
1
):
386
.
52.
Tekirdag
K
,
Cuervo
AM
.
Chaperone-mediated autophagy and endosomal microautophagy: joint by a chaperone
.
J Biol Chem
.
2018
;
293
(
15
):
5414
24
.
53.
José-Enériz
ES
,
Román-Gómez
J
,
Cordeu
L
,
Ballestar
E
,
Gárate
L
,
Andreu
EJ
, et al
.
BCR-ABL1-induced expression of HSPA8 promotes cell survival in chronic myeloid leukaemia
.
Br J Haematol
.
2008
;
142
(
4
):
571
82
.
54.
Ota
J
,
Yamashita
Y
,
Okawa
K
,
Kisanuki
H
,
Fujiwara
SI
,
Ishikawa
M
, et al
.
Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders
.
Oncogene
.
2003
;
22
(
36
):
5720
8
.
55.
Côté
S
,
Zhou
D
,
Bianchini
A
,
Nervi
C
,
Gallagher
RE
,
Miller
WH
.
Altered ligand binding and transcriptional regulation by mutations in the PML/RARα ligand-binding domain arising in retinoic acid–resistant patients with acute promyelocytic leukemia
.
Blood
.
2000
;
96
(
9
):
3200
8
.
56.
Su
M
,
Alonso
S
,
Jones
JW
,
Yu
J
,
Kane
MA
,
Jones
RJ
, et al
.
All-trans retinoic acid activity in acute myeloid leukemia: role of cytochrome P450 enzyme expression by the microenvironment
.
PLoS One
.
2015
;
10
(
6
):
e0127790
.
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