Abstract
Richter’s syndrome (RS) is the transformation of chronic lymphocytic
leukemia (CLL) into a high-grade B-cell malignancy. Molecular and
functional studies have pointed out that CLL cells are close to the
apoptotic threshold and dependent on BCL-2 for survival. However, it
remains undefined how evasion from apoptosis evolves during disease
transformation. Here, we employed functional and static approaches to
compare the regulation of mitochondrial apoptosis in CLL and RS. BH3
profiling of 17 CLL and 9 RS samples demonstrated that RS cells had
reduced apoptotic priming and lower BCL-2 dependence than CLL cells.
While a subset of RS was dependent on alternative anti-apoptotic
proteins and was sensitive to specific BH3 mimetics, other RS cases
harbored no specific anti-apoptotic addiction. Transcriptomics of
paired CLL/RS samples revealed downregulation of pro-apoptotic
sensitizers during disease transformation. Albeit expressed, effector
and activator members were less likely to colocalize with mitochondria
in RS compared to CLL. Electron microscopy highlighted reduced cristae
width in RS mitochondria, a condition further promoting apoptosis
resistance. Collectively, our data suggest that RS cells evolve
multiple mechanisms that lower the apoptotic priming and shift the
anti-apoptotic dependencies away from BCL-2, making direct targeting of
mitochondrial apoptosis more challenging after disease transformation.
graphic file with name 41419_2024_6707_Figa_HTML.jpg
Subject terms: Chronic lymphocytic leukaemia, Chronic lymphocytic
leukaemia
Introduction
Even in the modern treatment era, 2–10% of patients affected by chronic
lymphocytic leukemia (CLL) experience transformation into high-grade
histologies, most commonly into diffuse large B-cell lymphoma (DLBCL)
[[62]1]. This condition, known as Richter’s syndrome (RS), is
characterized by resistance to conventional and targeted agents, with a
median overall survival of less than 1 year [[63]1, [64]2]. Recently,
whole-genome sequencing and transcriptomics studies have identified
genes and pathways potentially driving RS [[65]3–[66]9]. These include
deletions of cell-cycle regulators, reduced expression of chromatin
remodelers [[67]3], point mutations of critical transcription factors
(TFs) [[68]3, [69]4, [70]9], and upregulation of gene sets fostering
mitochondrial oxidative metabolism [[71]3]. Despite the contribution of
multi-omics analyses, several functional aspects of RS biology have
remained unexplored due to the rarity of this disease and the limited
availability of live cell-based RS models, two conditions precluding
extensive ex vivo investigations.
Among the unexplored aspects of RS, the intrinsic (i.e., mitochondrial)
pathway of apoptosis appears of particular interest given its prominent
role in the regulation of life/death balance both in homeostasis and
during anti-cancer treatments. Intrinsic apoptosis is coordinated at
the outer mitochondrial membrane (OMM) by the BCL-2 family of proteins,
which are distinguished into anti-apoptotic members (BCL-2, MCL-1,
BCL-xL, BFL-1), pro-apoptotic effectors (BAK and BAX), BH3-only
activators (BIM, BID, PUMA) and sensitizers (BAD, BMF, HRK, NOXA)
[[72]10]. Mechanistically, anti-apoptotic proteins sequester activators
and sensitizers, preventing their interaction with BAK and BAX.
Following pro-apoptotic stimuli, activators are freed by sensitizers
and activate the effector members, which generate pores through the
OMM. This favors the leakage of cytochrome c (cyt c) from the
intermembrane space and the activation of cytosolic caspases [[73]11].
CLL cells typically express high levels of pro-apoptotic members that
are tonically sequestered by BCL-2, thereby having a high apoptotic
priming and a functional BCL-2 dependency [[74]12–[75]14].
Here, we used a series of live cell-based RS samples to investigate the
apoptotic priming and the anti-apoptotic dependencies of RS. We found
that RS cells lose the apoptotic priming and the BCL-2 dependency
typical of CLL, highlighting that indolent and aggressive disease
phases evade intrinsic apoptosis through divergent mechanisms.
Results
RS cells have reduced apoptotic priming
To gain insight into the evolution of apoptosis evasion during
high-grade transformation, we applied BH3 profiling on 17 CLL (9
treatment-naïve and 8 relapsed/refractory, Supplementary Table [76]1)
and 9 RS samples, and we set up a hypothesis-driven workflow to uncover
potential biological determinants of the different anti-apoptotic
profile of RS (Fig. [77]1A, Supplementary Fig. [78]1). RS cases
included 4 primary samples from RS patients in leukemic phase (RSCNO,
RSVR1, RSVR2, RSVR3), 4 RS-PDX models (RS9737, RS1050, IP867/17,
RS1316), and 1 RS cell line, U-RT1. Among RS primary samples
(Supplementary Fig. [79]2A), RSVR2 was consistent with RS-like
transformation due to abrupt ibrutinib interruption [[80]15, [81]16].
Only RSVR3 patient was treated with venetoclax prior to disease
transformation. Four promiscuous pro-apoptotic peptides (BIM, BID, BMF,
and PUMA) antagonizing all major anti-apoptotic proteins were applied
to permeabilized cells to interrogate the overall apoptotic priming.
Overall, RS samples had lower apoptotic priming than CLL cases (Fig.
[82]1B, C). The mean percentage of cyt c release upon incubation with
BIM, BID, PUMA and BMF was 97.2, 97.0, 97.5 and 96.4 for CLL versus
71.4, 60.3, 67.1, and 72.5 for RS, respectively (Fig. [83]1C, P <
0.0001 for each peptide). Of note, decreased apoptotic priming was
evident in RS but not in relapsed/refractory CLL samples (P < 0.001
when comparing relapsed/refractory CLL cases with RS samples),
indicating that high-grade transformation and not simply disease
relapse or TP53 mutational status (Supplementary Fig. [84]3) is
required to modulate the apoptotic priming. The median 90% effective
maximal concentration (EC[90]) of BIM, a putative biomarker of
chemosensitivity [[85]17], was 0.08 ± 0.09 μM for CLL and not reached
for all RS cases with the exception of RSVR3 (EC[90]: 0.73 μM; Fig.
[86]1D), consistent with the presence of highly unprimed cell
subpopulations within the RS tumor bulk. Notably, matched CLL-RS
samples longitudinally collected from two individual patients
(CLL#10/RSVR1 and CLL#14/RSVR2) clearly confirmed the loss of apoptotic
priming during disease transformation (Fig. [87]1E). In the RS-like
sample RSVR2, the apoptotic priming was reacquired after ibrutinib
resumption and concomitantly with the reappearance of the small-cell
morphology at the peripheral blood smear (Fig. [88]1E).
Fig. 1. Characterization of RS apoptotic priming.
[89]Fig. 1
[90]Open in a new tab
A Schematic of study design. Samples from 17 CLL patients and 9 RS
patients/models were subjected to intracellular BH3 profiling to derive
their apoptotic priming and anti-apoptotic dependencies. Then, a
multimodal approach was set up to explore why RS harbored a different
functional apoptotic profile as compared to CLL. Particularly, we
assessed the expression of BCL-2 family members at transcript and
protein level, the subcellular localization of pro-apoptotic effectors
and activators, and the width of mitochondrial cristae. B Heatmap of
the percentage of cyt c loss as quantified by flow cytometry on CLL and
RS samples (green, lowest value; red, highest value). Each column is a
sample, and each row refers to a BH3-only peptide with which cells were
incubated for 60 min. Circles and triangles underneath the heatmap
indicate matched CLL/RS samples longitudinally collected from two
individual patients. The Burkitt lymphoma cell line DG-75, not
expressing BAK and BAX and hence fully resistant to intrinsic apoptosis
[[91]54], was used as negative internal control for cyt c release. C
Percentage of cyt c release for each of the 17 CLL and 9 RS samples
upon incubation with the promiscuous peptide (BIM, BID, PUMA, or BMF)
indicated on the y axis of each graph. Unpaired Student t test;
means ± SEM. ****P < 0.0001. D Dose–response curves for BIM peptide.
EC[90] was 0.08 ± 0.09 μM for CLL and not reached for RS samples. E BH3
profiling of matched CLL/RS samples. CLL#10 developed DLBCL-type RS
(RSVR1) after 4 years of treatment with ibrutinib. CLL#14 developed
RS-like transformation (RSVR2) 1 week after abrupt ibrutinib
interruption. RSVR2 represents a transient state, as large leukemic B
cells reacquired the typical CLL morphology after ibrutinib resumption.
All the indicated peptides were used at 10 μM. Images underneath the
left graph refer to cytospin of CLL and RS PBMC. Those underneath the
right graph refer to peripheral blood smears at different timepoints.
Slides were stained with May-Grunwald Giemsa. Original magnification
x1000. Scale bar: 10 μm.
RS cells show loss of BCL-2 dependence
To interrogate functional anti-apoptotic dependencies, we incubated CLL
and RS cells with an array of BH3 peptides antagonizing specific
anti-apoptotic proteins (Fig. [92]1B, Supplementary Fig. [93]1A, B). We
found that RS cases were less BCL-2 dependent than CLL samples. The
mean cyt c release to BAD minus HRK peptide, a metric for BCL-2
dependence [[94]18], was 72.8% for CLL versus 18.8% for RS (Fig.
[95]2A, P < 0.0001). As with the overall priming, relapsed/refractory
status or TP53 mutations (Supplementary Fig. [96]3) were not sufficient
to drive the loss of BCL-2 dependence, which occurred only in
transformed disease (P < 0.0001 when comparing relapsed/refractory CLL
with RS). Incubation with the small molecule BCL-2 antagonist
venetoclax, used the same way as for BH3 peptides, produced similar
results with decreased cyt c release in RS (Fig. [97]2A). Kinetic
analysis of caspase 3/7 activation following venetoclax treatment
demonstrated induction of apoptosis in CLL cells as soon as 4 h after
venetoclax addition. In contrast, RS cells proved insensitive to
venetoclax within a 7-hour time frame (Fig. [98]2B), further indicating
their inferior vulnerability to BCL-2 antagonism. Apart from the RS1050
model, which expressed the lowest level of BCL2 transcript with no
detectable protein, all the other tested RS samples showed some degree
of BCL-2 expression that was in some case even higher than what
observed in CLL (Fig. [99]2C, D). This indicates that functional BCL-2
dependence in CLL and RS cannot be predicted solely on the basis of
BCL-2 expression.
Fig. 2. Reduced functional dependence upon BCL-2 in RS cells.
[100]Fig. 2
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A Percentage of cyt c release for each of the 17 CLL and 9 RS samples
upon incubation with selective peptides (BAD, HRK) or the BCL-2
antagonist venetoclax at the indicated concentrations. As regards
peptides, BCL-2 dependence was derived by subtracting the effect of HRK
(specific for BCL-xL) from that of BAD (interacting with both BCL-2 and
BCL-xL). Unpaired Student t test; means ± SEM. ****P < 0.0001;
***P < .001; **P < 0.01. B Real-time monitoring of caspase 3/7
activation upon incubation of 5 CLL and 5 RS samples (4 RS-PDX and
RSVR1) with 50 nM venetoclax for up to 7 hours. Whiskers: min to max
value. Unpaired Student t test. *P < 0.05. C qPCR analysis of BCL2 in
the indicated RS and CLL samples. Unpaired Student t test. *P < 0.05. D
Western blot analysis for BCL-2 in the indicated CLL and RS samples.
The dot plot represents intensity of proteins bands in CLL (open
circles) and RS (solid circles). Band intensities were measured using
Image Lab and normalized on Actin. Data are reported as mean ± SEM.
A subset of RS upregulates alternative anti-apoptotic dependencies
Having identified a reduced BCL-2 dependence in Richter’s cases, we
wondered if some of them upregulated alternative anti-apoptotic
dependencies. Out of 9 RS samples, five upregulated alternative
dependencies, also showing some degree of co-dependencies (Fig.
[102]3A). Particularly, RS1050 was co-dependent on MCL-1, BCL-xL, and
BFL-1, RS9737 was co-dependent on MCL-1 and BFL-1, whereas RSVR3, the
RS-like RSVR2 and RS1316 were mainly MCL-1 dependent. The RSCNO case
was the only one preserving both a relatively high apoptotic priming
and some degree of BCL-2 dependence. The remaining samples displayed
more marked loss of PUMA-triggered apoptotic priming and did not show
any specific anti-apoptotic dependency (Fig. [103]3A). Although CLL
samples were invariably characterized by high BCL-2 dependence, they
acquired co-dependence upon MCL-1 in the relapsed/refractory setting
(Fig. [104]3B). mRNA expression of the anti-apoptotic genes alternative
to BCL-2 was partially consistent with the functional data, with RS1050
showing the highest levels for MCL1, BCL2L1 (encoding BCL-xL) and
BCL2A1 (encoding BFL-1) (Fig. [105]3C). Although there was a trend
towards a higher expression of these transcripts in RS, the comparison
with CLL was not statistically significant. Western blot analysis
confirmed that RS1050 displayed the highest expression of BCL-xL and
BFL-1 even at the protein level (Fig. [106]3D). In addition, we
performed BH3 profiling using the selective BCL-xL antagonist
A-1331852, and found that RS1050 was the only one showing up to 42.3%
of cyt c release in response to this agent (Fig. [107]3E), indicating
that a subset of RS might be targeted by BH3 mimetics that antagonize
alternative anti-apoptotic proteins.
Fig. 3. Upregulation of alternative anti-apoptotic dependencies in a subset
of RS.
[108]Fig. 3
[109]Open in a new tab
BH3 profiling of the indicated RS (A) and CLL (B) samples. BCL-2
dependence was derived by subtracting the effect of HRK (specific for
BCL-xL) from that of BAD. MCL-1, BCL-xL, and BFL-1 dependencies were
derived by using MS-1, HRK, and FS-1 peptides, respectively, at the
concentration of 10 μM. BIM and PUMA were used at the concentration of
10 μM as well. Unpaired Student t-test. *P < 0.05. C qPCR analysis of
MCL1, BCL2L1 (encoding BCL-xL), and BCL2A1 (encoding BFL-1) in the
indicated CLL and RS samples. D Western blot analysis for MCL-1,
BCL-xL, and BFL-1 in CLL and RS samples. E BH3 profiling of the
indicated RS samples after incubation with the specific BCL-xL
antagonist A-1331852. n = 3.
Antagonism of non-BCL-2 anti-apoptotic dependencies and inhibition of
proximal signaling pathways are possible ways to trigger apoptosis in RS
As BH3 profiling revealed that a subgroup of RS harbors non-BCL-2
anti-apoptotic dependencies, we tested whether specific BCL-xL and
MCL-1 inhibitors could trigger apoptosis in intact RS cells. RS samples
were incubated with A-1331852 or [110]S63845 (a selective MCL-1
inhibitor) and caspase 3/7 activation was monitored over time. In
accordance with BH3 profiling, A-1331852 was effective only in RS1050,
while [111]S63845 was mostly effective in RS1316 and RSVR3 (Fig.
[112]4A), further suggesting that direct antagonism of BCL-xL and MCL-1
could rapidly promote apoptosis in disease models with such specific
anti-apoptotic dependencies. Because the transcription of pro-survival
genes alternative to BCL2 is often driven by upstream oncogenic
signaling [[113]19–[114]22], we also reasoned that rational inhibition
of key pathways could extinguish non-BCL-2 anti-apoptotic dependencies.
RSVR3 patient, a chemo-refractory RS (Supplementary Fig. [115]2)
characterized by the highest apoptotic priming among RS, high MCL-1
dependency (Figs. [116]3A, [117]4A) and active BTK signaling (Fig.
[118]4B), was treated with ibrutinib. Two weeks after ibrutinib
initiation, MCL-1 expression was reduced in leukemic RS cells (Fig.
[119]4C). Likewise, MCL-1 dependency decreased from 90.8% to 39.9%,
with only transient increase of BCL-2 dependency (Fig. [120]4D). While
on ibrutinib, the apoptotic priming remained high and no pro-survival
proteins were effectively preventing apoptosis. Such decreased
anti-apoptotic protection paralleled with a clear-cut reduction of
lactate dehydrogenase (LDH), slow disappearance of RS cells from
peripheral blood, and improved bone marrow function (Fig. [121]4E). To
investigate whether inhibition of key signaling pathways could increase
the apoptotic priming of RS samples, including those with no
anti-apoptotic dependency, we incubated RS-PDXs and U-RT1 cell line
with clinical-grade pathway inhibitors, chosen for their putative
anti-RS activity based on previous pre-clinical studies
[[122]23–[123]25]. We found that pathway inhibitors improved the
apoptotic priming in the majority of RS models (Fig. [124]4F).
Copanlisib, a PI3Kαδ inhibitor, was one of the most active compounds in
our screen and was able to augment the apoptotic priming even in
IP867/17 (Fig. [125]4F), which had no clear-cut anti-apoptotic
dependency at baseline (Fig. [126]3A). In addition to increasing the
overall priming, short term copanlisib treatment exposed multiple
anti-apoptotic dependencies (Fig. [127]4G) potentially exploitable for
selective targeting or combinatorial strategies. Collectively, these
data suggest that while RS cases harboring non-BCL-2 anti-apoptotic
dependencies could be targeted by direct or indirect suppression of the
participant anti-apoptotic member, those with no clear anti-apoptotic
dependency could be effectively primed (and re-sensitized to apoptosis
targeting) by acting on more proximal signaling pathways (Fig.
[128]4H).
Fig. 4. Antagonism of alternative anti-apoptotic dependencies and inhibition
of proximal signaling as possible ways to trigger apoptosis in RS.
[129]Fig. 4
[130]Open in a new tab
A Real-time monitoring of caspase 3/7 activation upon incubation of the
indicated RS samples with 50 nM A-1331852 (BCL-xL inhibitor, left
panel) or 5 μM [131]S63845 (MCL-1 inhibitor, right panel). As indicated
on the y axis, results are normalized on DMSO control for each specific
timepoint. n = 3. Data are reported as mean ± SEM. B Histograms of
fluorescence of pBTK^Tyr223 in leukemic RSVR3 cells. Upper-right inset
is the peripheral blood smear of RSVR3 patient showing two typical RS
cells characterized by large size, basophilic cytoplasm and one or more
nucleoli. Magnification: x1000. Scale bar: 10 μm. C Histograms of
fluorescence of MCL-1 in leukemic RSVR3 cells before and after
ibrutinib (Ibr) start. FMO: fluorescence minus one. D BH3 profiling of
RSVR3 before and after ibrutinib start. Apoptotic priming was derived
by using 10 μM BIM. BCL-2 dependence was derived by subtracting the
effect of HRK (specific for BCL-xL) from that of BAD, or by applying
0.1 μM venetoclax. MCL-1, BCL-xL, and BFL-1 dependencies were derived
by using MS-1, HRK, and FS-1 peptides, respectively, at the
concentration of 10 μM. E PBMC and platelet (PLT) count (left y axis),
and LDH values (U/L; right y axis) in RSVR3 before and after ibrutinib
initiation. F Dynamic BH3 profiling. RS samples were treated with the
indicated drugs or DMSO for 16 h. Afterwards, they were incubated with
BIM peptide. The heatmap shows the delta priming percentage values,
corresponding to the cyt c loss of drug-treated cells minus cyt c loss
of DMSO-treated cells. The higher the delta priming, the more effective
the drug is in increasing the apoptotic priming. G Extended dynamic BH3
profiling of RS1316 and IP867/17. Cells were treated with 1 μM
copanlisib for 3 and 6 h. Then they were subjected to BH3 profiling
using the indicated peptides at the concentration of 1 μM. Heatmaps
show that in RS1316 cells copanlisib treatment increased the MCL-1,
BCL-xL, and BFL-1 dependencies for up to 38%, 47 and 40% at 6 h,
respectively. In IP867/17, copanlisib increased MCL-1 and BCL-xL
dependencies for up to 38 and 81% at 6 h, respectively. H Schematic of
CLL and RS apoptotic profiles. CLL are characterized by high apoptotic
priming and high BCL-2 dependency, but relapsed/refractory cases can
acquire additional, non BCL-2, anti-apoptotic dependencies. In
contrast, RS cases are characterized by lower apoptotic priming and
BCL-2 dependency than CLL. Based on their BH3 profiling and sensitivity
to BCL-xL and MCL-1 inhibitors, RS cases can be further distinguished
into those with no clear anti-apoptotic dependency and those with at
least one anti-apoptotic dependency. While in the first subset only the
targeting of upstream pro-survival pathways can be exploited to
increase the apoptotic priming or expose specific anti-apoptotic
dependencies, in the latter direct or indirect pharmacological
inhibition of the participant pro-survival member can be devised as
effective therapeutic approach.
Selected pro-apoptotic sensitizers are transcriptionally downregulated in RS
To investigate whether decreased apoptotic priming could be driven by
transcriptional modulation of pro-apoptotic members, we performed
single-cell RNA sequencing on CLL#14 (CLL phase)/RSVR2 (RS-like phase)
longitudinal samples. A total of 7762 cells were analyzed, 6271 were
B-cells according to Garnett Classification (Supplementary Fig.
[132]4A). Gene set enrichment analysis (GSEA) pathway enrichment
analysis showed “positive regulation of apoptotic process” and
“positive regulation of cell death” among the top-20 downregulated
pathways in the RS-like phase. Conversely, pathways related to
structural components of intracellular organelles and regulation of
metabolic processes were strongly upregulated (Fig. [133]5A). Among
pro-apoptotic genes of the BCL-2 family, the sensitizers HRK and PMAIP1
(encoding NOXA) were the only ones significantly downmodulated in the
RS-like phase. By contrast, none of the pro-apoptotic effectors and
activators were modulated during large-cell transformation (Fig.
[134]5B). Cell fate trajectory analysis confirmed the acquisition of a
LDHA^high/HRK^low/PMAIP1^low state along pseudotime progression (Fig.
[135]5C), further highlighting the emergence of proliferative and
anti-apoptotic programs during disease transformation.
Fig. 5. Single-cell transcriptomics of matched CLL/RS samples.
[136]Fig. 5
[137]Open in a new tab
A Gene set enrichment analysis (GSEA) of the top 20 up- and
downmodulated pathways in the RS-like versus CLL phase. Only pathways
with >250 genes were considered. The normalized enriched score
represents the maximum deviation from zero. P < 0.001, FDR < 0.001. B
Fold change and statistical significance of the pro-apoptotic BCL-2
family genes in the RS-like versus CLL phase. FDR: false discovery
rate. C Cell fate trajectory analysis showing the pseudotime
progression of B cells and the expression of LDH, HRK and PMAIP1 along
with pseudotime progression. Upper panels: CLL phase. Lower panels:
RS-like phase. D Upper panel. Unsupervised clustering of B cells
(CLL + RS-like) showing 5 different clusters with the indicated BCL-2
family biomarkers. Lower panel. Cluster distribution in the CLL and
RS-like phase. E Heatmap illustrating the regulon expression pattern of
RS-like and CLL phase after applying SCENIC R-based package to our
dataset. Differentially expressed regulons are written on the right
side of the heatmap. F Alluvial plot showing the BCL-2 family genes
(Targets, on the right) that are controlled by the transcription
factors (TFs, on the left) identified by gene regulatory network
analysis. Blue and red lines refer to TFs enriched in CLL and RS-like
phase, respectively. Grey-highlighted BCL-2 family genes (HRK and
PMAIP1) are those differentially expressed in the two disease phases
(downregulated in RS-like cells).
To assess if different pro- and anti-apoptotic relatives are expressed
in distinct tumor subpopulations, we performed unsupervised clustering
of leukemic cells and searched for BCL-2 family genes among the
biomarkers of each cluster. Five different clusters were identified.
Cluster 4 was enriched in cells expressing HRK. Cluster 1 had a higher
BCL2A1 expression as compared to the others, whereas cluster 3
displayed higher BID expression (Fig. [138]5D, Supplementary Fig.
[139]4B). Interestingly, the HRK^high cluster underwent contraction
upon disease transformation, confirming the downmodulation of this
sensitizer in the RS-like phase (Fig. [140]5D).
To uncover differences in gene regulatory network (GRN) between CLL and
RS-like phase, we applied SCENIC workflow [[141]26] to our dataset and
identified 10 differentially expressed regulons. Each regulon
corresponds to an individual TF with its target gene set. Six regulons
were upregulated in the CLL phase (TCF4, JUND, ENO1, BCLAF1, SPIB, and
SMARCB1), and 4 in the RS-like phase (ZNF274, JUN, FLI1, and BACH2)
(Fig. [142]5E, Supplementary Fig. [143]4C). These GRN-based differences
enabled a clear segregation of CLL and RS-like timepoints
(Supplementary Fig. [144]4A). Importantly, the CLL-specific SPIB1,
BCLAF1, and SMARCB1 were identified as putative TFs activating HRK and
PMAIP1 expression in the CLL phase (Fig. [145]5F).
To evaluate if the loss of pro-apoptotic sensitizers could be a
recurrent theme during high-grade evolution, we manually interrogated
two publicly available RNA sequencing datasets from matched CLL-RS
samples [[146]3, [147]4]. The dataset from Nadeu and colleagues
included single-cell results from 4 CLL patients transformed into
DLBCL-type RS. Three and 2 out of 4 patients downregulated PMAIP1 and
HRK during disease transformation, respectively. Paradoxically, the
pro-apoptotic activators BCL2L11 (encoding BIM) and BID were more
likely to be upregulated rather than suppressed during RS, and the
effector BAX showed a heterogeneous pattern of modulation across
patients (Supplementary Fig. [148]4D). Dataset from Parry and
colleagues, which included bulk RNA sequencing results from 5 cases,
showed an overall downregulation of pro-apoptotic sensitizers in RS,
with BMF being the only one reaching statistical significance
(Supplementary Fig. [149]4E). Additionally, the pro-apoptotic
sensitizer NOXA was nearly absent at the protein level in our series of
RS-PDXs (Supplementary Fig. [150]4F).
Because transcriptional deregulation of PUMA drives acquired resistance
to venetoclax in CLL [[151]27], we assessed its expression in indolent
and aggressive disease phases. Our transcriptomic analysis together
with Nadeu’s and Parry’s data showed no difference in terms of BBC3
(PUMA) expression between CLL and RS (Fig. [152]5B, Supplementary Fig.
[153]4D, E). qPCR and western blot revealed similar PUMA expression in
our RS models and CLL samples as well (Supplementary Fig. [154]5A, B).
Immunohistochemical assessment of 8 CLL and 4 RS biopsies, including 3
matched CLL-RS samples, showed an unexpectedly higher expression of
PUMA in RS compared to CLL (Supplementary Fig. [155]5C), suggesting
that CLL cases acquiring resistance to BCL-2 antagonism [[156]27] and
those transforming into RS might modulate PUMA in an opposite manner.
Altogether, high-grade transformation is accompanied by transcriptional
downregulation of selected pro-apoptotic sensitizers, while leaving
unchanged or even paradoxically upregulated the pro-apoptotic effectors
and activators.
Pro-apoptotic effectors and activators are less likely to colocalize with
mitochondria in RS
Western blot analysis showed heterogeneous expression of BAK, BAX, and
BID in RS samples, with no significant difference when compared to CLL.
Moreover, the activator BIM was even more expressed in RS with respect
to CLL (Fig. [157]6A). During homeostasis, part of these molecules
localizes at the OMM and part is constrained within the cytosol. The
ratio between their cytosolic and mitochondrial fraction inversely
correlates with chemosensitivity in human cancers [[158]28, [159]29].
To evaluate whether subcellular localization of effectors and
activators differ between CLL and RS, we stained these pro-apoptotic
members with specific monoclonal antibodies and used fluorescence
microscopy to quantify the proportion that colocalized with
mitochondria. Colocalization coefficient was automatically calculated
for each Z-stack section of randomly selected CLL and RS cells. In line
with previous data, BAK had a higher degree of colocalization with
mitochondria than BAX in both disease conditions [[160]30].
Importantly, while BAK displayed similar colocalization coefficients in
CLL and RS cells (P = .11), BAX, BIM and BID were less likely to
colocalize with mitochondria in RS as compared to CLL (P < 0.0001; Fig.
[161]6B–E), suggesting high-grade cells need a second hit to bring
these pro-apoptotic molecules onto the mitochondria for apoptosis
induction.
Fig. 6. Subcellular localization of selected pro-apoptotic effectors and
activators in CLL and RS.
[162]Fig. 6
[163]Open in a new tab
A Western blot analysis for the indicated pro-apoptotic effectors and
activators in CLL and RS. Dot plots represent intensity of proteins
bands in CLL (open circles) and RS (solid circles). Band intensities
were measured using Image Lab and normalized on Actin. Data are
reported as mean ± SEM. Unpaired Student t-test. *P < .05. (B-E) Four
primary CLL samples and 3 RS-PDX models (RS9737, RS1316, and RS1050)
were subjected to fluorescence microscopy to analyze the subcellular
localization of pro-apoptotic effectors and activators. Each sample was
stained with MitoTracker Deep Red FM to identify mitochondria, and with
fluorescent monoclonal antibodies specific for the indicated
pro-apoptotic proteins: B BAK, C BAX, D BIM, E BID. Samples were
counterstained with DAPI. After selecting the field of interest (red
circle), colocalization analysis was performed using Costes
thresholding strategy automatically provided by the Zen module.
Colocalization coefficients (=number of pixels colocalized with
MitoTracker/total number of pixels occupied by the pro-apoptotic
protein) of each Z-stack section of 8 CLL cells (2 for each patient)
and 8 RS cells (3 for RS9737 and RS1316, and 2 for RS1050) were plotted
(BAK: CLL n = 258 and RS n = 351; BAX: CLL n = 399 and RS n = 215; BIM:
CLL n = 257 and RS n = 270; BID: CLL n = 273 and RS n = 155). Whiskers
represent 10–90 percentile. Unpaired Student t-test, ****P < .0001;
***P < .001. Scale bar: 2 μm.
RS mitochondria exhibit reduced cristae width
Cyt c is retained within the mitochondrial intermembrane space until
pro-apoptotic stimuli reach the threshold for OMM permeabilization. The
width of mitochondrial cristae has been demonstrated as an additional
regulator of intrinsic apoptosis [[164]31–[165]33]. While wide cristae
allow rapid cyt c leakage once OMM is permeabilized, tight cristae
entrap cyt c within the intermembrane space, thus hindering downstream
pro-apoptotic events [[166]33]. Given the different propensity to
release cyt c, we hypothesized that CLL and RS mitochondria could
harbor different cristae conformations. To test this, we performed TEM
on 4 CLL samples and 4 RS-PDX models and focused the analysis on
mitochondrial morphometry. Compared to CLL, RS cells had a lower number
of mitochondria per surface unit, with no differences in terms of
diameter (Fig. [167]7A). Importantly, the width of mitochondrial
cristae was reduced in RS cells with respect to CLL cells (mean cristae
width: 11.7 ± 0.26 nm in CLL versus 9.41 ± 0.17 nm in RS, P < 0.0001;
Fig. [168]7B). Expression of OPA1 and CLPB, two mitochondrial proteins
involved in cristae tightening [[169]31–[170]33], was evaluated by
western blot. While OPA1 levels did not change between CLL and RS, CLPB
was more expressed in RS (Fig. [171]7C). This was consistent with qPCR
results showing higher CLPB transcript in RS (Fig. [172]7D). These
findings suggest the acquisition by RS cells of a mitochondrial
morphology that further favors apoptosis evasion.
Fig. 7. Ultrastructure of CLL and RS mitochondria.
[173]Fig. 7
[174]Open in a new tab
A Representative electron micrographs of CLL and RS cells for each
analyzed sample. Original magnification: ×8900. Scale bar: 500 nm.
Mitochondria were manually counted in 19 CLL (5 cells for #1, 5 for #6,
5 for #8, and 4 for #9) and 20 RS cells (5 cells for each model), and
results were represented as number of mitochondria divided by cell
area. Unpaired Student t test; means ± SEM. **P < .01. Mitochondrial
diameter (minor axis length) was also measured in 191 and 200
mitochondria from the CLL and RS groups, respectively. B Representative
electron micrographs of mitochondria from the indicated CLL and RS
samples. Black arrowheads indicate examples of mitochondrial cristae in
CLL and RS cells. Maximal cristae width was quantified on micrographs
of 50 randomly selected mitochondria from 14 CLL and 14 RS cells
(n = 141 cristae each group). Original magnification: x56000. Scale
bar: 100 nm. Unpaired Student t-test; means ± SEM. ****P < 0.0001. C
Western blot analysis for OPA1 and CLPB in CLL and RS samples. Dot
plots represent intensity of proteins bands in CLL (open circles) and
RS (solid circles). Band intensities were measured using Image Lab and
normalized on Actin. Data are reported as mean ± SEM. *P < 0.05. D qPCR
analysis of CLPB in the indicated RS and CLL samples. Unpaired Student
t-test. *P < .05.
Discussion
Through a comparative analysis of apoptosis regulation in CLL and RS,
we provide evidence that the anti-apoptotic strategies adopted by
leukemic cells evolve during disease transformation. RS mitochondria
acquired the ability to retain cyt c even when exposed to high amounts
of pro-apoptotic peptides, thereby having a low apoptotic priming, and
are less dependent upon BCL-2 for survival as compared to CLL. In some
RS cases (e.g., RSVR3, RS1050, RS1316), MCL-1, BCL-xL, and BFL-1
exerted a prominent role in protecting cancer cells from apoptosis,
suggesting their direct targeting might be effective and outperform
BCL-2 antagonism. Conversely, other RS samples (e.g., U-RT1, IP867/17)
did not harbor any specific anti-apoptotic dependency and did not
undergo apoptosis following incubation with MCL-1 or BCL-xL
antagonists. For these samples, inhibition of upstream oncogenic
pathways could be a valuable approach to increase the apoptotic priming
and to timely expose anti-apoptotic dependencies for effective
combination strategies. Single-cell transcriptomics of a matched CLL-RS
case together with results from two independent studies [[175]3,
[176]4] indicated the class of pro-apoptotic sensitizers, particularly
HRK and NOXA, was recurrently downregulated at transcriptional level
during high-grade transformation. Of note, epigenetic suppression of
HRK has been implicated in the pathogenesis of several human cancers
including high-grade lymphomas [[177]34, [178]35], and NOXA has been
identified as a rheostat for venetoclax sensitivity in different types
of blood malignancies [[179]36–[180]38]. Surprisingly, we found no
evidence of downregulation for pro-apoptotic effectors and activators,
which were in some cases even more expressed in RS compared to CLL.
This may suggest for these molecules some tumor-beneficial,
non-apoptotic roles ultimately fostering proliferative programs and
metabolic rewiring [[181]39, [182]40].
Despite having comparable expression in CLL and RS, BAX as well as BIM
and BID displayed a lower level of colocalization with mitochondria in
RS. Moreover, decreased apoptotic priming of RS cells parallels the
tightening of their mitochondrial cristae. The shape of mitochondrial
cristae has long been recognized as a key determinant of mitochondrial
function, with narrow cristae favoring the assembly and stability of
respiratory chain complexes [[183]41]. Functional and transcriptomic
data are piling up about the upregulation of oxidative phosphorylation
in RS [[184]3], a metabolic signature that may find in cristae
tightening its morphological correlate. While in vitro manipulation of
cristae width has been shown to tune the apoptotic priming of cancer
cells [[185]33], our work is the first one demonstrating that changes
in cristae shape may naturally occur during high-grade transformation,
a condition eventually furthering the metabolic efficiency and the
anti-apoptotic attitude of RS mitochondria.
Our functional results are in line with the clinical results of BCL-2
antagonism. Objective responses have been observed in 75% of CLL
patients treated with venetoclax as single agent, with a median
progression-free survival of 30.2 months [[186]42]. In contrast, only 3
out of 7 RS patients were sensitive to venetoclax in the phase I
first-in-human study, and only in one patient the duration of response
exceeded 24 months [[187]43]. Though disappointing as monotherapy in
RS, venetoclax deserves further exploration in combination with agents
that increase the apoptotic priming and the BCL-2 dependence of RS
cells, such as chemotherapy and BTK inhibitors [[188]44, [189]45].
Moving beyond clinical observations, the reduced apoptotic priming of
RS can be ultimately considered as a large step away from B-cell
physiology. Several immunology studies indicate that fully functional
intrinsic apoptosis is required to delete autoreactive clones during
B-cell ontogenesis and eliminate activated B cells producing
low-affinity antibodies [[190]46–[191]48]. Indeed, normal lymphocytes
usually have a high apoptotic priming [[192]49]. From this viewpoint,
CLL cells appear strikingly like their normal counterpart, still
preserving an effective commitment to apoptosis that is only controlled
by the function of one, or at most two, anti-apoptotic proteins
[[193]12]. This relatively simple way to evade apoptosis is nowadays
successfully beaten by venetoclax. In sharp contrast, RS cells
implement a series of mechanisms, including downmodulation of
sensitizers, cytosolic relocation of pro-apoptotic members, cristae
tightening, and upmodulation of heterogeneous non-BCL-2 anti-apoptotic
dependencies, that render apoptosis evasion more robust in nature and
hence much harder to dismantle.
A potential limitation of our study is that all primary RS samples had
leukemic involvement by large B cells and were collected from
peripheral blood. Although this allowed a fairer comparison with CLL
samples, collected from peripheral blood as well, we acknowledge that
the majority of RS cases encountered in the clinic have no leukemic
spread and manifest as fast-growing nodal or extranodal masses.
Moreover, one of our primary samples was compatible with RS-like
transformation, a transient state triggered by abrupt ibrutinib
interruption and pathogenetically distinct from true RS. Therefore,
additional studies involving RS cells collected from tumor masses are
needed to validate and expand our findings.
In conclusion, our work reveals a functional shift away from apoptosis
commitment during high-grade transformation, defining poor apoptotic
priming and low BCL-2 dependency as two novel functional hallmarks of
RS. Moreover, we have identified multiple mechanisms potentially
involved in apoptotic priming collapse. A broader understanding of
these processes might open the way to more rational strategies for
directly targeting apoptosis in RS and possibly other types of
aggressive lymphomas.
Methods
Patient samples and RS models
The study was undertaken in accordance with the principles of the
Declaration of Helsinki and was approved by the local ethics committee
(protocol #1747CESC). All enrolled patients provided written informed
consent. Seventeen clinically and biologically annotated CLL samples
and 4 RS samples in leukemic phase were obtained from the University
Hospital of Verona and from the Hematology Unit, Hospital S. Croce e
Carle, Cuneo. All CLL patients fulfilled the current criteria for CLL
diagnosis [[194]50]. RS diagnosis was always confirmed by bone marrow
or lymph node histology. All primary RS and RS models were clonally
related to prior CLL. In all primary RS cases enrolled in this study,
large B cells were detected at peripheral blood smear and collected for
downstream analyses. Large B cells represented 80%, 68%, 85% and 65% of
the total leukemic cells in RSVR1, RSVR2, RSVR3, and RSCNO,
respectively. RS-like syndrome induced by ibrutinib interruption
(RSVR2) was defined by the appearance of large B cells in peripheral
blood, accompanied by clinical (B symptoms) and laboratory signs
(LDH > 2 times of upper normal limit) of disease transformation.
Peripheral blood mononuclear cells (PBMC) were viably frozen in fetal
bovine serum (FBS) supplemented with 10% DMSO until thawing. PBMC from
CLL patients were composed for more than 90% of leukemic cells. Four
previously characterized RS-PDX models, maintaining the same phenotypic
and genetic features of the primary tumors, were utilized [[195]51,
[196]52]. Three of them (RS9737, RS1316, and RS1050) were established
from patients who had received chemotherapy and/or targeted agents,
whereas the fourth (IP867/17) was established from a treatment-naïve
patient. The local animal care and use committee approved this study.
Mice were treated according to the European guidelines for animal use
in scientific research, and with the approval of the Italian Ministry
of Health (authorization #664/2020; protocol #CC652.136). For in vitro
experiments, RS cells were withdrawn from tumor masses of PDX models
and cryopreserved until use. U-RT1 [[197]53] and DG-75 [[198]54] (DSMZ,
Braunschweig, DE) cell lines were cultured in RPMI supplemented with
10% FBS. Cell lines were authenticated routinely and Mycoplasma free.
BH3 profiling and dynamic BH3 profiling
BH3 profiling was chosen over other methods that investigate
anti-apoptotic dependencies due to its short incubation time. CLL and
RS samples have different rates of spontaneous apoptosis ex vivo.
Therefore, cytotoxicity results derived from prolonged incubation with
small molecules are biased by the different proclivity to commit
spontaneous apoptosis across samples in culture. BH3 profiling was
performed as previously described by Ryan and colleagues [[199]18],
with minor modifications. Thawed cells were resuspended at a
concentration of 2 × 10^6/mL in PBS/Zombie Yellow viability dye (1:500)
(BioLegend, San Diego, CA) for 30 min at room temperature (RT). After
washing with PBS/2% FBS, cells were resuspended in the same buffer and
stained for 15 min RT with anti-human CD5-FITC and anti-human CD19-V450
(Becton Dickinson, San Jose, CA) monoclonal antibodies. After washing
in PBS, cells were resuspended in MEB2 buffer (150 mM mannitol, 10 mM
HEPES, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% IgG-free BSA, pH 7,5 with
KOH) at 4 × 10^6/mL. To prepare BH3 profiling reactions, 100 μL of
MEB2/20 μg/mL digitonin was added to flow cytometry tubes. Synthetic
peptides (Peptide Synthetics, Fareham, UK) or small molecules
(venetoclax or A-1331852, from Selleckchem, Munich, DE) were added at
different concentrations to each tube and 100 μL of cell suspension was
dispensed in each tube, in duplicate. Tubes were incubated at 25 °C in
the dark in gentle agitation for 60 min. Cells were fixed for 10 min by
the addition of 67 μL PBS/4% formaldehyde methanol-free (Thermo Fisher
Scientific, Waltham, MA), quenched for 15 min by 67 μL N2 buffer (1.7 M
Tris base, 1.25 M glycine, pH 9.1) and stained overnight at 4 °C with
40 μL of 10X intracellular staining buffer (10% BSA, 2% Tween20 in PBS)
containing anti-human cytochrome c-Alexa Fluor 647 monoclonal antibody
(1:400, BioLegend). A total of 30,000 CD5^+/CD19^+ cells were acquired
on a FACSCantoII cytometer (Becton Dickinson) and analyzed by FlowJo
9.9.6 software (Tree Star, Ashland, OR). Hierarchical gating was used
to accurately identify cells of interest and to exclude doublets and
dead cells (Supplementary Fig. [200]1). Following morphological gate
(FSC-A/SSC-A) and doublets exclusion (FSC-A/FSC-H), the remaining dead
cells were excluded by gating on Zombie Yellow negative cells. In
primary samples, the population of interest was identified by the
immunological gate on CD5^+/CD19^+ cells. In RS-PDX models, the
population of interest was identified by the immunological gate on
CD19^+ cells. Because RS cells did not harbor specific surface markers
and morphological details were lost upon digitonin exposure, large RS
cells could not be separated from residual CLL cells during the BH3
profiling analysis of primary samples. An inert peptide (PUMA2A) was
used to define full cyt c retention (negative control) while 25 μM
alamethicin (Abcam, Cambridge, UK), served as a complete cyt c release
control (positive control). The effect of peptides and drugs was
calculated by using median fluorescence intensity of the cyt c
staining, as follows:
[MATH: %cytcloss=MFIsample-<
/mstyle>MFIalamethicin/MFIPUMA2A-MFIalamethicin :MATH]
Dose–response curves for BIM peptide were generated with GraphPad Prism
9 using log(inhibitor) vs. response–variable slope (four parameters).
The concentrations of BH3 peptides used for the generation of the
heatmap in Fig. [201]1B are detailed in Supplementary Table [202]2.
For dynamic BH3 profiling (DBP) [[203]55], cells were plated in
RPMI-1640/penicillin-streptomycin/10% FCS and treated with a panel of 9
pathways inhibitors, each at the concentration of 1 μM for 16 h. Cells
were collected, washed in PBS, and subjected to BH3 profiling using BIM
(0.005 μM for RS1050 and RS9737, 0.05 μM for RS1316 and IP867, and 1 μM
for U-RT1) or PUMA2A peptides. The concentration of BIM was chosen to
obtain a basal (non-drug induced) cyt c release ranging from 10 to 30%.
To calculate the percent change in mitochondrial priming (delta
priming), we first determined the percentage of cyt c loss for cells
exposed to pro-apoptotic peptides alone as for standard BH3 profiling.
Finally, the following formula was applied:
[MATH: delta
priming=%cyt c
lossdrug−%cyt c
lossDMSO :MATH]
For the extended DBP shown in Fig. [204]4G, the indicated RS-PDX models
were treated with 1 μM copanlisib for 3 or 6 h and then subjected to
BH3 profiling using BAD, MS-1, HRK, or FS-1 peptides, each at the
concentration of 1 μM.
Real-time monitoring of caspase 3/7 activation
Induction of apoptosis upon venetoclax treatment was monitored over
time using the CellEvent^TM Caspase-3/7 Green Assay Kit (Thermo Fischer
Scientific). Regarding the experiment in Fig. [205]2B, PBMC from CLL
#6, #7, #9, #13 and RS cells from each of the RS-PDX models were
dispensed in polylysine-coated 24-well plates at a density of 10^6
cells/well, and treated with DMSO or 50 nM venetoclax. For the
experiments shown in Fig. [206]4A, RS models were treated with DMSO, 50
nM A-1331852 or 5 μM [207]S63845. Kinetics of Caspase-3/7 cleavage was
evaluated by time-lapse video microcopy with a Zeiss AxioObserver 7
inverted wide-field microscope, equipped with thermostatic chamber,
Colibri 7 fluorescent LED illumination, full motorized stage, Hamamatsu
ORCA-Flash4.0 V3 Digital CMOS camera, set at 8 output bit depth, and
the Zeiss ZEN 2.6 time-lapse module (Oberkochen, DE). Plates were kept
at 37 °C in 5% CO[2] humidified atmosphere; movie acquisition was with
a 40x Plan Apochromatic objective (AN 0,65) corresponding to an
acquisition area of 1.2 × 10^5 mm^2, with a cell density of 500
cell/acquisition area. Each field was acquired in brightfield and
fluorescent light illumination (503/530 nm ex/em). Exposure time for
fluorescent light was set at 500 msec and left unchanged for the entire
duration of the experiment. Time-lapse imaging was for 7 h, with a
30 min time frame. Frame-by-frame image analysis was performed, without
image preprocessing, with the Zeiss ZEN 2.6 image analysis module,
allowing automatic cell segmentation, recognition and pixel intensity
quantification. Data were expressed and plotted as normalized number of
Caspase-3/7 positive cells over time.
Single-cell RNA sequencing of patient undergoing RS-like transformation
PBMC from an individual patient in two different disease phases (CLL
#14 and the RS-like phase RSVR2) were isolated and viably frozen. After
thawing, cells were resuspended in RPMI supplemented with 5% FBS to
achieve a final concentration of 1000 cells/mL. Ten thousand live cells
were loaded onto the Chromium controller to recover 4000 single-cell
GEMs per inlet uniquely barcoded. After cDNA synthesis, sequencing
libraries were generated. Final 10× library quality was evaluated using
the Fragment Analyzer High Sensitivity NGS kit (Agilent Technologies,
Santa Clara, CA) and then sequenced on the Illumina NextSeq500
(Illumina, San Diego CA) generating 75 base pair paired-end reads
(28 bp read1 and 91 bp read2) at a depth of 50,000 reads/cell. Raw base
call (BCL) files were processed using Cell Ranger (10× Genomics) from
PartekFlow software to obtain a unique molecular identifier (UMI) count
table. To perform these steps, Homo Sapiens (Human) reference data
(hg38 Feb 3, 2022) was downloaded from the 10× official website. Next,
the dataset was analyzed using the PartekFlow software. As first, we
filtered out low-quality cells, such as doublets, damaged cells, or
those with too few reads, evaluating the number of read counts per cell
(600–15000), the number of detected genes per cell (200–4000), the
percentage of mitochondrial reads per cell (0–10), and the percentage
of ribosomal counts per cell (10–60) After quality control, we obtained
a total of 7762 cells. Following the recommended normalization step,
through which counts were normalized and presented in logarithmic scale
in CPM (count per million) approach, features were filtered excluding
genes that are expressed by any cells in the dataset, thus obtaining a
total number of 15296 genes. Batch correction was provided using the
general linear model task, available in the PartekFlow. Garnett
Classification was used to identify B cells (n = 6271), on which UMAP
dimensional reduction and unsupervised clustering were performed.
Clusters biomarkers were automatically computed by the software
performing a Student’s t-test on the selected attribute, comparing one
subgroup at a time with all the others combined. Gene-specific analysis
(GSA) and GSEA were performed on the cell population of interest using
PartekFlow plugins. Similarly, we used PartekFlow software to run,
after the scaling expression, the trajectory analysis, based on
Monocle3 R package. We thus identified states and branch points, also
calculating pseudotime values. Study of gene regulatory network was
performed by applying SCENIC R-based package to our dataset [[208]26].
Western Blots
Primary CLL (10–20 × 10^6), RS-PDX (2 × 10^6), and U-RT1 (2 × 10^6)
cells were lysed and protein concentration measured using the Bradford
Protein Assay (Bio-Rad, Milan, Italy), according to the manufacturer’s
protocol. Proteins were resolved using a 12% MiniPROTEAN® TGX™ Precast
Protein Gels (Bio-Rad) and transferred into 0.2 μm nitrocellulose
Trans-Blot Turbo Transfer membrane using the Trans-Blot Turbo Transfer
System (all from Bio-Rad). Membranes were blocked with 5% non-fat dry
milk in a Tris buffer 0.1% Tween prior to primary antibody incubation
(overnight; 4 °C) followed by a secondary antibody HRP-conjugated. All
the antibodies used are listed in Supplementary Table [209]3. Blots
were incubated with the Clarity or Clarity Max Western ECL substrates
and images acquired with a ChemiDoc XPS+ imaging system (Bio-Rad).
Densitometric quantification was performed using the Image Lab Bio-Rad
software and bands were normalized on the β-actin, used as a loading
control. Original blots are provided in Supplementary Fig. [210]6.
Real-time quantitative polymerase chain reaction (qPCR)
RNA was extracted from primary CLL (1–10 × 10^6), RS-PDX (5 × 10^5),
and U-RT1 (5 × 10^5) cells using the RNeasy Plus Mini Kit (Qiagen,
Milan, Italy), and retro-transcribed to cDNA using the High-Capacity
cDNA Reverse Transcription Kit (Applied Biosystems Thermo Fisher,
Milan, Italy), according to the manufacturer’s instructions. qPCR was
performed using 4 ng cDNA with iTaq Universal Probes SuperMix
(#1725134, BioRad) and gene-specific probes for B2M (Hs00984230_m1),
BCL-2 (Hs00608023_m1), BCL-xL (Hs00236329_m1), MCL-1 (Hs01050896_m1),
BBC3 (Hs00248075_m1), CLPB (Hs00229376_m1), BCL2A1 (Hs00187845_m1), all
from Thermo Fisher Scientific using the CFX384 Real-Time System
(Bio-Rad). Reactions were performed in triplicate. For each gene,
expression levels were computed as a ratio of the number of copies of
the target gene over 10^5 copies of B2M.
Flow cytometry assessment of BTK activation and MCL-1 expression
BTK activation was measured by means of tyrosine 223 phosphorylation.
Briefly, 0.5 × 10^6 cells were fixed in 100 μL 4% formaldehyde for
30 min at 4 °C. Cells were washed in PBS and suspended in 50 μL of
permeabilization buffer (PBS + 5% FBS + 0.5% saponin) containing
PE-conjugated anti-pY223-BTK antibody (BD Biosciences) or isotype
control (Merck, Rahway, NJ) for 30 min at 4 °C. Cells were washed and
suspended in 200 μL ice-cold PBS and analyzed by flow cytometry. For
MCL-1 expression, 1 × 10^6 cells were stained with Fixable Viability
Stain 780 (BD, 5665388) in PBS for 15 min at room temperature. After
washing cells in Facs Buffer (PBS1X + 2%FBS) and staining with
CD19-PE-Cy7 (SJ25C1 clone, 557835, BD) for 15 min at RT, cells were
washed with permeabilization buffer (00-5523, eBioscience) and fixed in
100 μL 0.4% formaldehyde for 30 min at 4 °C. Intracellular staining was
performed by using anti-Mcl-1 Alexa-Fluor 488-conjugated antibody
(D2W9E clone, BK583265 CST, Euroclone) in permeabilization buffer for
30 min at 4 °C. Cells were washed in permeabilization buffer and
analyzed by flow cytometry.
PUMA immunohistochemistry
Tissue sections from formalin-fixed paraffin-embedded (FFPE) primary RS
biopsies and CLL-RS matched sample biopsies were stained with an
anti-PUMA rabbit polyclonal antibody (ab9645, Abcam, Cambridge, UK),
followed by an anti-rabbit HRP-conjugated antibody and
3,3’-diaminobenzidine (EnVision™ System, Dako, Glostrup, DK) to
visualize the reaction. Anti-PUMA specificity on tissues was tested on
reactive lymph nodes and breast cancer samples. Immunohistochemical
expression of PUMA was evaluated semiquantitatively as percentage of
positive cells (0%; 0–25%; 25–50%; 50–75%: >75%) and as staining
intensity (1+, 2+, 3+).
Fluorescence microscopy and colocalization analysis
CLL and RS-PDX cells were stained with 300 nM MitoTracker Deep Red FM
(Thermo Fisher Scientific) for 30 min at 37 °C. Cells were fixed with
formaldehyde 0.4% for 20 min and washed with PBS/5% FCS. Cells were
permeabilized with PBS/0.1%Triton X-100 for 7 min at 4 °C. After
washing and blocking for 15 min at 4 °C with PBS/5% FCS, cells were
incubated for 1 h at 4 °C with mouse anti-human BAX and BID (BD
Biosciences, Franklin Lakes, NJ and Proteintech, Rosemont, IL,
respectively) and rabbit anti-human BAK and BIM (Abcam) monoclonal
antibodies. After washing, cells were incubated for 40 min at 4 °C with
goat anti-mouse or goat anti-rabbit IgG AlexaFluor488 antibodies
(Abcam). After washing, cells were stained with 1 μg/mL DAPI,
transferred onto a glass slide, and mounted with Fluoro Gel with DABCO
(Electro Microscopy Sciences, Hatfield, PA). Images were acquired with
a wide field Zeiss AxioImager Z.2 deconvolution microscopy setting,
equipped with Colibri 7 fluorescent LED illumination, motorized 3D
scanning stage, and Hamamatsu ORCA-Flash4.0 V3 Digital CMOS camera, set
at 8 bit output depth. 512 × 512 pixel ROIs were acquired with a 100x
Plan Apochromatic oil immersion objective (AN 1.46). Each field was
acquired with triple fluorescent light illumination (385/30 nm ex. for
DAPI, 475/36 nm ex. for Alexa Fluor 488 and 631/33 nm ex. for Cy5-Deep
Red FM). Automatic 3D image scanning was according to the
Nyquist-Shannon sampling theorem, by using the inline ZEN 3.5 Nyquist
Calculator. 3D scans were, then, processed with Zeiss ZEN 3.5 by
applying the advanced Zeiss Deconvolution (DCV) module. Image
deconvolution was achieved by applying the Constrain Iterative
algorithm, without auto-normalization to fully control the photon
budget thus allowing full reassignment of photons from out-of-focus
optical planes. Spectral linear unmixing was, finally, applied to
remove overlapped spectral components and background noise. Deconvolved
and unmixed 3D stacks were rendered and analyzed with the ZEN 3.5
Colocalization module. Colocalization of pro-apoptotic proteins with
mitochondria was quantified in each slice using Costes thresholding
strategy [[211]56], automatically provided by the Zen module.
Colocalization coefficients (calculated as ratio of number of pixels
colocalized with Mitotracker over total number of pixels occupied by
the pro-apoptotic protein) of each section of 8 CLL cells from 4
individual patients and 8 RS cells from 3 RS-PDX models (RS9737,
RS1316, RS1050) were plotted.
Transmission electron microscopy
For ultrastructural examination, cell pellets of 4 CLL patients (#1,
#6, #8, and #9) and 4 RS-PDX models were fixed for 1 h in 2%
glutaraldehyde in 0.1 M phosphate buffer and, after washing, postfixed
for 1 h in 1% OsO[4] diluted in 0.2 M K[3]Fe. Subsequently, samples
were dehydrated in graded concentrations of acetone and embedded in a
mixture of Epon and Araldite (Electron Microscopic Sciences, Fort
Washington, PA). Ultrathin sections were cut at 70 nm thickness on an
Ultracut-E ultramicrotome (Reichert-Jung), stained with lead citrate,
and observed on a Philips Morgagni 268 D transmission electron
microscope operating at 80 kV (Fei Company, Eindhoven, NL), equipped
with Megaview II camera for acquisition of digital images. The
mitochondrial count was performed on micrographs at ×5600 of 19
randomly selected CLL cells (5 cells from #1, 5 from #6, 5 from #8, and
4 from #9) and 20 randomly selected RS cells (5 for each sample). The
minor axis length was measured on micrographs at ×8900 of 191 CLL and
200 RS mitochondria from 20 different cells. The cristae width was
measured on micrographs at x56000 of 50 randomly selected mitochondria
from 14 cells in the CLL group and 50 mitochondria from 14 cells in the
RS group, for a total of 141 cristae each condition. Measurements were
taken using Emsis Radius 2 software and are expressed as mean ± SEM.
Statistics
Statistical analyses were conducted using GraphPad Prism 9. Unpaired
two-tailed Student t test was used to compare unpaired groups. Unless
otherwise specified, data are presented as means ± SEM. Differences
were considered significant for P values <0.05.
Supplementary information
[212]Supplementary information file^ (1.3MB, pdf)
Acknowledgements