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\begin{document}

\title{Repurposing of thalidomide and its derivatives for the treatment of
SARS-coV-2 infections: Hints on molecular action}



\author[1]{Lakshmikirupa Sundaresan}%
\author[2]{Suvendu Giri}%
\author[2]{Himanshi Singh}%
\author[2]{Suvro Chatterjee}%
\affil[1]{Hospital for Sick Children}%
\affil[2]{AU-KBC Research Centre}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: SARS-coV-2 pandemic continues to cause an
unprecedented global destabilization. There is an urgent need to develop
vaccines or identify molecules to treat severe cases and repurposing of
drugs is the best approach at this hour. Thalidomide, despite having an
infamous history has been successfully repurposed and tested for various
disease conditions including inflammatory diseases and tumor. Few
reports emphasize the use of thalidomide with a SARS-coV-2 pneumonia
patient being successfully treated with thalidomide. Experimental
Approach: A meta-analysis comparing the transcriptomes of SARS-coV-2
infected tissues with thalidomide and lenalidomide-induced
transcriptomic changes in transformed lung, endothelial and
hematopoietic models was performed. Key Results: Thalidomide and
lenalidomide exhibited pleiotropic effects affecting a range of
biological processes including inflammation, immune response,
angiogenesis, MAPK signaling, NOD-like receptor signaling, TLR
signaling, leukocyte differentiation and innate immunity, the processes
which are aberrantly regulated in severe COVID-19 patients. In addition,
we show the similarities between the expression profiles of SARS-coV-2
infected lung and systemic lupus erythematous. Conclusion and
Implications: The present study recommends thalidomide analogs as a
``better fit'' to treat severe cases of novel viral infections, healing
the damaged network by compensating the impairment caused by the
Coronavirus disease-2019 (COVID-19).%
\end{abstract}%



\sloppy


\textbf{Repurposing of thalidomide and its derivatives for the treatment
of SARS-coV-2 infections: Hints on molecular action}

Running title: Possible mechanisms of thalidomide in treating COVID-19

Lakshmikirupa Sunderesan\selectlanguage{ngerman}\textsuperscript{¥}, Suvendu
Giri\textsuperscript{\#,*}, Himanshi Singh\textsuperscript{\#,*}, and
Suvro Chatterjee\textsuperscript{\#,*}

*Vascular Biology Laboratory, AU-KBC Research Centre, Chennai, India.

\textsuperscript{\#}Department of Biotechnology, Anna University,
Chennai, India.

\textsuperscript{¥}Developmental~\& Stem Cell Biology Program, The
Hospital for Sick Children, Toronto, ON M5G 1X8,~Canada

\textbf{Correspondence:}

Suvro Chatterjee Ph.D,

AU-KBC Research Centre,

MIT Campus of Anna University,

Chromepet, Chennai-600044, India.

Tel.: +91 44 2223 4885×48/+91 44 2223 2711×48;

Fax: +91 44 2223 1034

E-mail address: soovro@yahoo.ca

\textbf{Word count} : 2,733

\textbf{Acknowledgement} : This project was partially supported by a
grant from University Grant Commission-Faculty Recharge Programme
(UGC-FRP), Government of India to SC.

\textbf{Conflict of Interest} : The authors declare that there are none.

\textbf{Abstract}

\textbf{Background and Purpose} : SARS-coV-2 pandemic continues to cause
an unprecedented global destabilization. There is an urgent need to
develop vaccines or identify molecules to treat severe cases and
repurposing of drugs is the best approach at this hour. Thalidomide,
despite having an infamous history has been successfully repurposed and
tested for various disease conditions including inflammatory diseases
and tumor. Few reports emphasize the use of thalidomide with a
SARS-coV-2 pneumonia patient being successfully treated with
thalidomide.

\textbf{Experimental Approach:} A meta-analysis comparing the
transcriptomes of SARS-coV-2 infected tissues with thalidomide and
lenalidomide-induced transcriptomic changes in transformed lung,
endothelial and hematopoietic models was performed.

\textbf{Key Results:} Thalidomide and lenalidomide exhibited pleiotropic
effects affecting a range of biological processes including
inflammation, immune response, angiogenesis, MAPK signaling, NOD-like
receptor signaling, TLR signaling, leukocyte differentiation and innate
immunity, the processes which are aberrantly regulated in severe
COVID-19 patients. In addition, we show the similarities between the
expression profiles of SARS-coV-2 infected lung and systemic lupus
erythematous.

\textbf{Conclusion and Implications:} The present study recommends
thalidomide analogs as a ``better fit'' to treat severe cases of novel
viral infections, healing the damaged network by compensating the
impairment caused by the Coronavirus disease-2019 (COVID-19).

\textbf{Keywords:} SARS-coV-2, Thalidomide, Lenalidomide, CC-220,
Pomalidomide, Inflammation, Immune Response, Angiogenesis

\textbf{Abbreviations} : COVID-19 - Coronavirus Disease -19; BALF --
Broncho-alveolar lavage fluid; PBMC -- Peripheral Blood Mononuclear
Cell; DEG- Differentially Expressed Genes; GSEA -- Gene Set Enrichment
Analysis; SLE -- Systemic Lupus Erythematosus; TLR -- Toll-like
Receptor; HUVEC -- Human Umbilical Vein Endothelial Cell

\textbf{Introduction}

Novel coronavirus, SARS-coV-2 has been posing devastating effects on a
global scale with a soaring number of infections and an alarming rate of
mortality. Despite tremendous efforts, development of effective vaccines
or anti-viral medications is months away leaving repurposing of drugs
with known safety and efficacy profiles as the only viable choice.
COVID-19 is clinically very challenging since the novel coronavirus
triggers multi-organ turbulence devastating the homeostasis of the human
system. Once the SARS-CoV-2 virus enters the respiratory tract, there
are four different stages of the infection from symptoms to multi-organ
failures. Phase I starts with the ACE2 receptor-mediated viral entry
through nasal and oral routes followed by host immune system alert
simultaneously with active viral replication in the upper respiratory
tract (Phase II). Next, Phase III coincides with minor cytokine storm in
the alveoli releasing the inflammatory cytokines making the blood
vessels leaky around the air sac, and ultimately the second cytokine
storm arrives with uncontrolled inflammatory and life-threatening
symptoms, acute respiratory distress syndrome (ARDS), seizure, severe
hypoxia and severed organ toxicity (Phase IV) . Manifestation of the
bi-phasic cytokine storm occurs through the activation of a series of
cytokines including granulocyte-colony stimulating factor (G-CSF),
interferon gamma-induced protein 10 (CXCL10), monocyte chemoattractant
protein 1 (MCP1), macrophage inflammatory protein 1\selectlanguage{greek}α \selectlanguage{english}(MIP-1\selectlanguage{greek}α\selectlanguage{english}), tumour
necrosis factor \selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}), interleukin (IL)-2R and IL-6 overwhelming the
system leading to indiscriminate damages in multiple organs . There is
an increased amount of blood vessel growth in the lungs of COVID-19
patients compared to severe influenza . Due to such multi-layers of
problems associated with the COVID-19 infections, researchers around the
world are desperately in search for a drug, which would able to tackle
all or few of these COVID-19 hallmarks.

Thalidomide, a small molecule drug, was marketed with an intention to
relieve morning sickness in pregnant women, and took a wrong turn that
resulted in children with birth defects upon in-utero exposure . Later,
thalidomide became a game changer for its the multi-faced
pharmacological effects such as immunomodulation, anti-inflammation,
anti-angiogenesis, and anti-viral effects . At this point of time, the
world needs a ``smart'' solution. Therefore, thalidomide raises the hope
for treating COVID-19 patients Chen et al reports successful treatment
of SARS-coV-2 associated pneumonia with combinatory treatment of
thalidomide and a low-dose glucocorticoid Two clinical trials,
NCT04273581 and NCT04273529 have been registered to check the efficacy
of thalidomide in treating COVID-19 patients. The adverse effects of
thalidomide and its analogs are well documented. Various genes
aberrantly expressed in SARS-coV-2 affected lungs are known targets of
thalidomide (Table S1). Extensive information available on thalidomide's
mechanisms, their efficacy and safety in hemophagocytic syndrome-induced
cytokine storm and idiopathic pulmonary fibrosis , severe H1N1 and
paraquat poisoning lung injury argue for the possible action of
thalidomide on COVID-19 induced lung effects and cytokine storm.

Transcriptome-based approach to connect diseases with drug responses is
a recognized strategy in drug repurposing . With the fast-growing
literature on SARS-coV-2 infections, we performed meta-analysis of whole
transcriptome signatures of lungs, PBMC, BALF from SARS-coV-2 affected
patients and A549 cells and compared with the gene expression signatures
of thalidomide or lenalidomide-treated A549 (transformed adenocarcinoma
cells), hematopoietic and endothelial cells. We hereby provide possible
mechanistic actions of thalidomide in treating the SARS-coV-2 pathology.
In addition, we suggest that the derivatives of thalidomide,
lenalidomide and CC-220 might also be effective in the treatment of
SARS-coV-2.

\textbf{Methods}

\textbf{Data Collection}

A total of 16 gene expression datasets including 15 publicly available
expression datasets were meta-analyzed in this study. Transcriptomes of
SARS-coV-2 infected lung tissues matched with healthy control and
SARS-coV-2 treated A549 cells were obtained from GEO (Accession ID
GSE147507). Transcriptome data of bronchoalveolar lavage fluid (BALF)
and peripheral blood mononuclear cells (PBMC) were obtained from the
recently published study . Differentially expressed genes (DEG) from the
expression profiles of systemic lupus erythematosus (Accession ID:
GSE112087), bone marrow cells treated with lenalidomide (Accession ID:
GSE106748), lymphoma cells treated with lenalidomide (Accession ID:
GSE60618), CD34 positive cells treated with pomalidomide (Accession ID:
GSE144052), MERS infected PBMC (Accession: GSE1739), lenalidomide
treated PBMC (Accession ID: GSE84251) and CC-122 treated lymphoma cells
(Accession ID: GSE75420) were obtained using limma from the GEO
database. Library of Integrated Network-Based Cellular Signatures
(iLINCS) is a database which contains the gene expression signatures of
more than 21,000 compounds (http://www.ilincs.org/ilincs/). We obtained
the gene expression signatures for A549 cells treated with 10\selectlanguage{greek}µ\selectlanguage{english}M
thalidomide for 6h (LINCSCP\_4683) and 24h (LINCSCP\_4463), 100 \selectlanguage{greek}µ\selectlanguage{english}M
lenalidomide for 6h (LINCSCP\_4650) and 24h (LINCSCP\_4427).

\textbf{Transcriptome sequencing}

SARS-coV-2 affects the cardiovascular system to a great extent and to
understand the effects of thalidomide and its derivatives, human
umbilical vein endothelial cells (HUVEC) were subjected to 20\selectlanguage{greek}µ\selectlanguage{english}M
thalidomide or 20\selectlanguage{greek}µ\selectlanguage{english}M lenalidomide or 20\selectlanguage{greek}µ\selectlanguage{english}M pomalidomide or vehicle control
treatment for 8h. RNA was isolated using TRIzol\selectlanguage{ngerman}® method and whole
transcriptome sequencing was performed using Illumina HiSeq 2500
platform. The data can be accessed at Gene Expression Omnibus (GEO) with
the Accession ID: GSE118979. The sequence reads were aligned with
reference genome of \emph{Homo sapiens} using TopHat2 (v2.0.8) and the
followed by transcript compilation and gene identification was done
using Cufflinks (v2.2.0) . The DEG were identified using Cuffdiff
program (v2.2.0)

\textbf{Differential Expression and Enrichment Analysis}

The raw counts from the SARS-coV-2 transcriptomic profiles were
subjected to differential expression analysis by DESEq2 v1.26.0 .
Subsequently the genes were pre-ranked using the p-values from DESeq2
analysis and subjected to pre-ranked gene set enrichment analysis (GSEA)
. Gene sets with FDR \textless{}0.05 were considered to be statistically
significant and were visualized using EnrichmentMap plugin of Cytoscape
. For drug signatures from iLINCS, differentially expressed genes with
p\textless{}0.05 were considered to be statistically significant.
Enrichment of kinase perturbation was carried out using Enrichr .

\textbf{KINOMEscan Kinase Screening}

We analyzed our previously published KINOMEscan kinase screening dataset
of thalidomide in order to investigate how thalidomide affects the
immune system. Kinases whose activities were reduced at least by 60\%
were considered for further enrichment analysis.

\textbf{Comparative analysis using Toppcluster}

The differentially expressed genes (Q\textless{}0.05 for transcriptome
and p\textless{}0.05 for drug signatures) from all the gene expression
profiles were compared for overlapping genes and over-represented
pathways using Toppcluster and the networks were visualized using in
Cytoscape .

\textbf{Identification of protein targets using PharmMapper}

For identifying the protein targets of the thalidomide, we utilized the
PharmMapper server (http://www.lilab-ecust.cn/pharmmapper/) . The server
identifies possible physiological protein targets of any drug molecule
by using a pharmacophore-based mapping approach. The three-dimensional
structure of thalidomide was obtained from PubChem and processed on the
PharmMapper server choosing only human protein target sets. The top 100
target proteins were selected based on the ranking associated with a fit
score (pKd value) for further enrichment analysis using Enrichr .

\textbf{Results}

\textbf{Meta-analysis of SARS-coV-2 affected lung biopsies and PBMCs
reveal enrichment of various pathways pertaining to immune response}

SARS-coV-2 infection caused a massive surge in inflammatory response,
cytokine production and cytokine-mediated signaling. There was a
substantial up-regulation of immune response including the processes of
hematopoietic development and lymphocyte activation (Figure 1A,3A,3B).
Activation of viral life cycle and anti-viral interferon signaling was
observed in infected lungs and A549 cells (Figure 1B, 5).
Over-representation of pathways including NOD-like receptor signaling,
MAPK cascade, Measles and Influenza-A were seen in the infected lung as
identified by gene set enrichment analysis (Figure 1A). Targets of
transcription factors, ISRE and IRF were upregulated in SARS-coV-2
affected lungs similar to up-regulation observed in the PBMC of SLE
patients (Figure 1B, 1C). Enrichr analysis revealed that the genes
up-regulated in SARS-coV-2 lung were down-regulated when SYK was knocked
down or inhibited (Figure 1D). Expression to kinase (X2K) analysis
showed the possible perturbation of various MAP kinases, ABL1 and JNK1
(Figure 1E,1F). Human phenotype enrichment analysis shows
thrombocytopenia, poor wound healing, abnormality of lymphatic system,
serositis and abnormal anticoagulant pathways in SARS-coV-2 infected
lungs (Figure S1).

\textbf{Effect of Thalidomide on Kinases Implicated in Immune Response
and MAPK signaling}

The kinase screening assay identified key kinases involved in the
regulation of immune response are functionally modulated by thalidomide.
The most kinase affected was LCK and SYK (Figure 2C), critical
modulators of T cell receptor signaling. Many LCK substrates, SPI1,
TBK1, FOXP3, EGR1, ESR1, IRF1, CBL and STAT1 as well as SYK
phosphorylation targets such as OAS1 and MX1 were up-regulated in
SARS-coV-2 lung. Various processes mediating immune response including
JUN phosphorylation, IKappaB phosphorylation, JAK-STAT pathway,
leukocyte mediated immunity, neutrophil degranulation and activation, B
cell receptor signaling and MAPK cascade were found to be affected by
thalidomide (Figure 2D). PharmMapper results showed strong affinity for
LCK, HCK and SYK along with other proteins involved in innate and
adaptive immune response (Table S2, S3, S4). We studied the effects of
thalidomide and its derivatives on endothelium and identified the
down-regulation of several angiogenic genes (Figure S5 and Table S1).

\textbf{Up-regulated pathways and GO Biological Processes in SARS-coV-2
infection suppressed by thalidomide and lenalidomide}

SARS-coV-2 infected lungs, PBMC and A549 cells showed significant
upregulation of expression of genes involved in inflammation, cytokine
signaling, MAPK signaling and activation of cells mediating the immune
response whereas BALF exhibited a slightly different immune profile
where were leukocyte and neutrophil activation was suppressed (Figure
3A). Comparison of differentially expressed genes of all the signatures
yielded interesting results. Many of the processes up-regulated in
SARS-coV-2 infected tissues were suppressed by thalidomide and
lenalidomide in A549 cells and endothelial cells (Figure 2A,2B,3A).
Thalidomide-treated A549 cells showed suppression of key genes including
SYK, JUN, PIK3CA and HLA genes implicated in immune response (Figure 3B,
3C). Thalidomide and lenalidomide down-regulated various
pro-inflammatory and angiogenic genes aberrantly expressed in SARS-cov-2
infected lungs including CCL2 and TSC22D3 which are NF-\selectlanguage{greek}κ\selectlanguage{english}B modulators in
A549 cells (Figure S3, S4). Thalidomide and lenalidomide treatment
resulted in significantly suppression of cytokine response,
angiogenesis, inflammation, Fc Epsilon receptor signaling and MAPK
cascade (Figure 2A). In addition, lenalidomide down-regulated STAT1
expression, leukocyte differentiation, TLR signaling as well as IRF
activation (Figure 2B, 4B). Many genes implicated in NOD-like receptor
signaling overexpressed in SARS-coV-2 were suppressed by lenalidomide in
A549 and lymphoma cells (Figure 4C). B cell receptor signaling was
activated in SARS-coV-2 affected PBMC whereas T cell activation was
observed in SARS-coV-2 lungs. Translation of viral mRNA was exclusively
observed in SARS-coV-2 infected BALF whereas genes implicated in viral
entry and life cycle were up-regulated in SARS-coV-2 infected lungs,
BALF and A549 cells. Genes involved in viral entry and type I interferon
signaling were down-regulated in thalidomide-treated A549 cells and
lenalidomide-treated lymphoma, A549 and HUVEC (Figure 5A, 5C).

\textbf{Similarity of SARS-coV-2 expression profile with that of SLE,
lymphoma and multiple myeloma}

Comparative enrichment analysis of gene expression profiles for
disease-specific phenotypes revealed the similarity of SARS-infected
tissues with pneumonia, influenza, lymphoma, systemic lupus
erythematosus (SLE), multiple myeloma, asthma, auto-immune diseases,
asthma, pneumonia, and atherosclerosis while SARS-coV-2 PBMC showed
exclusive overlap with lymphoma, multiple myeloma and chronic
lymphocytic leukemia (CLL) (Figure 6A). The gene expression profile of
SARS-coV-2 infected lungs was very similar to that of PBMC from SLE
patients (Figure 6C). There was a significant overlap between SARS-coV-2
and SLE in up-regulated genes involved in immune regulation (Figure 3B),
interferon signaling (Figure 5B) and disease-specific phenotypes (Figure
6B, 6D, 6E). Many genes deregulated in lymphoma and multiple myeloma
were up-regulated in SARS-coV-2 infections as well (Figure 6B, 6E).
Several aberrantly expressed genes involved in these disease phenotypes
were down-regulated in thalidomide and lenalidomide treated A549 and
endothelium (Figure 6).

\textbf{Discussion}

SARS-coV-2 infection causes surge in a number of pathways related to
inflammation, cytokine signaling, leukocyte and lymphocyte activation,
innate and adaptive immune response marking the phenomenon of ``cytokine
storm''. As the whole immune system is affected during the SARS-coV-2
infection, immunomodulators would be highly beneficial in treating the
symptoms. A COVID-19 patient with pneumonia was treated successfully
with thalidomide and low dose glucocorticoid. There was a significant
decrease in the inflammatory cytokines including IL1-, IL-6 and IFN-\selectlanguage{greek}ϒ
\selectlanguage{english}and increase in the CD4+ and CD8+ T cells and NK cells. Thalidomide
reduced the severity of many COVID-19 symptoms such as lung lesions,
exudation due to its pleiotropic effects on the human system .
Hemophagocytic syndrome, a hyperinflammatory disorder is also another
condition in which cytokine storm occurs. It is frequently present with
extranodal natural killer/T cell lymphoma (ENKTL). Thalidomide was
effective in suppressing the cytokine storm through inhibition of NF-\selectlanguage{greek}κ\selectlanguage{english}B
based transcription of IFN-\selectlanguage{greek}ϒ \selectlanguage{english}and TNF genes and thalidomide along with
P-Gemox was highly effective in treating ENKTL patients in a Phase II
clinical trial . Comparison of SARS-coV-2 expression profiles with drug
signatures through enrichment analysis revealed striking actions of
thalidomide and lenalidomide in A549 and endothelial cells. The results
suggest that thalidomide and lenalidomide could reverse the devastating
effects of SARS-coV-2 infections on immune system. We selected A549, an
adenocarcinomic human alveolar basal epithelial cell line to test our
hypothesis that thalidomide would be effective against the cytokine
storms. The A549 cell line is an appropriate model for testing cytokine
storm targeting drugs since a previous study established this model by
infecting the cells with influenza A/H1N1 virus (PR-8) or nonstructural
protein 1 (NS1) plasmid to test the mechanisms behind inflammatory
cytokines/chemokines mediated ``cytokine storm'' Studies have utilized
A549 cells to show the effects of thalidomide on lung fibrosis . A
limitation of this study is that only 978 genes called ``landmark
genes'' are profiled in the iLINCS drug signatures. However, the
profiles are highly reproducible and represent the whole transcriptome .
Our models of A549 and HUVEC effectively capture the effects of
thalidomide in lungs as well as endothelium.

It is also emerging that SARS-coV-2 infections perturb vascular plexus
significantly and there is a substantial increase in the growth of new
blood vessels and evidence of intussusceptive angiogenesis with
overexpression of angiogenesis and hypoxia genes in the lungs of
COVID-19 patients . Cytokine storm and atherosclerosis are tightly
connected in SARS-coV-2 which is consistent with our analysis revealing
the enrichment of atherosclerosis in the SARS-coV-2 signatures (Figure
6). Thalidomide is a renowned modulator of vascular system, and it is
known to transcriptionally or functionally target various genes (Table
S1) up-regulated genes in the lungs of COVID-19 patients . As SARS-coV-2
infection has a huge impact on the hematopoietic system affecting the
myeloid cell maturation, we meta-analyzed the effects of thalidomide and
its derivatives on PBMC, bone marrow cells as well as lymphoma cells.
Thalidomide and lenalidomide exhibited attenuation of cytokine signaling
and inflammation in addition to its anti-angiogenic action (Figure 3A).
The drugs affected most of the pathways up-regulated in SARS-coV-2
affected lungs and PBMC (Figure 1A, 3A) in A549 cells, mandating direct
investigations in SARS-coV-2 infected models.

COVID-19 coincides with a strong neuro-endocrine modulation because the
disease devastates functions of the organs, and naturally the reciprocal
communication between the organs of the endocrine stress system gets a
set-back . ACE2 is expressed along the hypothalamus, pituitary and
adrenal (HPA) axis which is implicated in the stress response and
adrenal glands has the highest concentration of virus particles next to
lung . A high expression of ACE2 in brain is believed to be the reason
for the possible infection of the central nervous system in SARS
patients . Chronic elevated stress levels has been reported in SARS and
SARS-coV-2 patients even long after the outbreak . Notably, thalidomide
is also known for its neuro-endocrine modulation properties. Thalidomide
modulates CNS by reducing the generation of pro-inflammatory cytokines
such as IL-1, IL-6, IL-8 and TNF-\selectlanguage{greek}α \selectlanguage{english}through NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibition . There was
a down-regulation of genes involved in circadian wake cycle (Figure 1B,
S2) including PER3 in the PBMC of COVID-19 patients hinting on the
possible sleep disturbances in SARS-coV-2 patients. Thalidomide being a
well-known antiemetic and sedative action on the neuroendocrine axis
would relax the patients which is supported by the report that
thalidomide was effective in treating the anxiety and digestive symptoms
in the COVID-19 patient .

The anti-inflammatory properties of thalidomide and its analogs through
reduction of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}expression and NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibition are well
established . SARS-coV-2 infections showing elevated NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling and
rampage activation of immune response. Unlike other RNA viruses,
SARS-coV-2 suppresses TNF receptor-associated factors 3 (TRAF3)
activation, inhibiting NF-\selectlanguage{greek}κ\selectlanguage{english}B and IRFs, leading to suppression of early
pro-inflammatory and antiviral responses. Whereas later stages of the
infection show an enhanced expression of IRF targets in the lungs with
an activation of IL-1, IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}expression and inhibition of type
I interferon signaling . Activation of IRF and ISRE transcriptional
targets in SARS-coV-2 affected lungs is in agreement with previous
studies reporting the SARS biology . Thalidomide inhibited LCK activity
affecting STAT1 phosphorylation, cytokine mediated signaling, NF- \selectlanguage{greek}κ\selectlanguage{english}B
signaling, osteoclast differentiation and MAPK signaling through
modulation of various upstream activators and downstream effectors.
Lenalidomide, in addition, suppressed leukocyte differentiation, TLR
signaling along with IRF activation in A549 and lymphoma cells. The
effects of thalidomide and lenalidomide observed in our study are
consistent with the previous studies where thalidomide and lenalidomide
has been shown to inhibit IRF and STAT1 phosphorylation resulting in the
downregulation of interferon expression and TLR signaling .

The expression profile of SARS-coV-2 infected lungs, PBMC as well as
A549 cells show resemblance with profiles of lymphoma, multiple myeloma
and SLE. Therefore, drugs which are effective in treating SLE, lymphoma
and multiple myeloma might be effective against SARS-coV-2 infection.
Thalidomide and its derivatives show impressive efficacy in treating
multiple myeloma and certain forms of lymphoma . Remarkably,
hydroxychloroquine, an FDA approved SLE drug is currently being used in
the management of critically ill SARS-coV-2 patients . CC-220, another
thalidomide analog shows very promising results in phase I/II clinical
trials against SLE . CC-220 through suppression of~Ikaros and Aiolos
expression , transcription factors which are essential for
differentiation of leukocyte and NK cells thus modulating the innate
immune system. As innate immune system pathways are deregulated in
SARS-coV-2 infected lung and PBMC, further studies are warranted to
investigate the efficacy and safety of CC-220 in treating COVID-19

Any treatment strategy with thalidomide and its analogs including
repurposing thalidomide for COVID-19, should consider
thalidomide-induced adverse effects including neuropathy and venous
thromboembolism (VTE) . There have been many reports on COVID-19
patients develop blood clots , a dangerous issue which might be
aggravated with the use of thalidomide and lenalidomide. In addition,
lenalidomide might cause cytokine release syndrome in chronic
lymphocytic leukemia patient . Therefore, a very careful dosage regimen
has to be followed with all these drugs as serious adverse effects have
been observed during dose escalation earlier.

\textbf{Conclusion}

Our study sheds light on the possible mechanisms through which
thalidomide and lenalidomide might be effective in the management of
SARS-coV-2 pathology. Thalidomide and derivatives effectively modulating
various aberrantly regulated pathways infections with abundant
pharmacological information available make them promising candidates for
the treatment of novel coronavirus infections.

\textbf{References}

\textbf{Figure legends}

Figure 1: Characteristics of SARS-coV-2 infected lungs and A549 cells.
Gene Set Enrichment Analysis (GSEA) of genes modulated in A) SARS-coV-2
infected lung and B) A549 cells. C) Transcription factor enrichment of
genes up-regulated in SARS-coV-2 infection. D) Genes activated in
SARS-coV-2 overlapping with genes down-regulated upon kinase
perturbations. Up-regulated genes mapped to their transcription factors
and kinases in E) SARS-coV-2 lung and F) A549 cells.

Figure 2: Effect of Thalidomide and Lenalidomide on Immune System. GSEA
network of gene expression profiles of A) thalidomide and B)
lenalidomide treated A549 cells. C) Activities of kinase involved in
immunomodulation and MAPK signaling in the presence of thalidomide D)
Biological Pathways enriched by kinases affected by thalidomide.

Figure 3: A) GO Biological Process enrichment comparison of gene
expression signatures B) SARS-coV-2 infected tissues B) Overexpression
of immune response genes in SARS-coV-2 tissues and suppression by
thalidomide and lenalidomide C) B cell receptor signaling activated in
PBMC of SARS-coV-2 patient and T cell activation in SARS-coV-2 lungs D)
Modulation of ERK1 and ERK2 cascade in SARS-coV2 lung and thalidomide in
A549.

Figure 4: Activation of A) inflammation, B) TLR signaling, C) NOD-like
signaling, D) Osteoblast differentiation and E) Cytokine signaling in
SARS-coV-2 and genes targeted by thalidomide and lenalidomide in the
signaling pathways.

Figure 5: Viral life cycle and defense response to virus. A)
Up-regulation of pathways pertaining to viral entry, life cycle and
antiviral response was observed in SARS-coV-2 infected lungs, PBMC, BALF
and A549 cells and their modulation by thalidomide and lenalidomide.
Overexpression of genes implicated in B) Interferon signaling and C)
Type I interferon signaling in SARS-coV-2 infected lung and A549.

Figure 6: A) Similarity of SARS-coV-2 signatures with disease-specific
phenotypes. Differentially modulated genes in SARS-coV-2 and thalidomide
treatment similar to profiles of B) lymphoma and D) Multiple Myeloma. C)
Overlapping of SARS-coV-2 expression profile of SARS-coV-2 affected lung
with systemic lupus erythematosus.\selectlanguage{english}
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