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\begin{document}

\title{Functional Consequence and Therapeutic Targeting of Cryptic ALK Fusions
(ALK fus ) in Monosomy7 AML}



\author[1]{Makia K. Manselle}%
\author[1]{Rhonda E. Ries}%
\author[1]{Tiffany Hylkema}%
\author[1]{Amanda Leonti}%
\author[1]{Danielle C. Kirkey}%
\author[1]{Scott  Furlan }%
\author[1]{Soheil Meshinchi}%
\affil[1]{Fred Hutchinson Cancer Research Center}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Acute Myeloid Leukemia (AML) patients have a wide array of cytogenetic
and molecular aberrations which can influence response to therapy.
Monosomy7 (Mono7) is a rare subset within pediatric AML (prevalence of
4-5\%), that is highly associated with poor outcomes. Fusions involving
the \emph{ALK} gene (14.3\%) were exclusively identified within this
high-risk cohort while absent across all other AML. Given the dismal
outcomes of Mono7, we evaluated the use of crizotinib, an FDA-approved
tyrosine kinase inhibitor, used to treat patients with ALK fusions. Our
findings suggest that crizotinib may serve as a novel therapy for these
patients.%
\end{abstract}%



\sloppy


\emph{Functional Consequence and Therapeutic Targeting of Cryptic ALK
Fusions (ALK\textsuperscript{fus}) in Monosomy7 AML}

\textbf{Makia K. Manselle} \textsuperscript{1, 2}, Rhonda E.
Ries\textsuperscript{1}, MA Tiffany Hylkema\textsuperscript{1}, Amanda
Leonti\textsuperscript{1}, MS, Danielle C. Kirkey, MD\textsuperscript{1,
2} Scott N. Furlan, MD\textsuperscript{1}, and Soheil Meshinchi, MD,
PhD\textsuperscript{1, 2}

1 - Fred Hutchinson Cancer Center, Seattle, WA

2 - University of Washington, Seattle, WA

Correspondence to:

Makia Manselle

Fred Hutchinson Cancer Center

1100 Fairview Ave N, D2-345

Seattle, WA, 98109

(206) 667- 6474

\emph{mmansell@fredhutch.org}

Text word count: 1152

Abstract word count: 95

Running Title: ``Targeting Cryptic ALK fusions in Mono7''

Key words: AML, Cancer Genetics, Chemotherapy, Cytogenetics,
Hematology/Oncology

Figures included: 2

Tables: 1\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Abbreviation: & Phrase:\tabularnewline
\midrule
\endhead
Mono7 & Monosomy 7\tabularnewline
AML & Acute Myeloid Leukemia\tabularnewline
TpAML & Target Pediatric AML\tabularnewline
ALK & Anaplastic Tyrosine Kinase\tabularnewline
ALK\textsuperscript{fus} & ALK fusion(s)\tabularnewline
NSCLC & Non-Small Cell Lung Cancer\tabularnewline
ALCL & Anaplastic Large-Cell Lymphoma\tabularnewline
COG & Children's Oncology Group\tabularnewline
\bottomrule
\end{longtable}

\emph{Abstract:}

Acute Myeloid Leukemia (AML) patients have a wide array of cytogenetic
and molecular aberrations which can influence response to therapy.
Monosomy7 (Mono7) is a rare subset within pediatric AML (prevalence of
4-5\%), that is highly associated with poor outcomes. Fusions involving
the \emph{ALK} gene (14.3\%) were exclusively identified within this
high-risk cohort while absent across all other AML. Given the dismal
outcomes of Mono7, we evaluated the use of crizotinib, an FDA-approved
tyrosine kinase inhibitor, used to treat patients with ALK fusions. Our
findings suggest that crizotinib may serve as a novel therapy for these
patients.

\emph{1 - Introduction:}

Acute Myeloid Leukemia (AML) is characterized by a malignancy of cells
in the myeloid lineage and caused by diverse genomic alterations,
resulting in a heterogenous disease with varied response to conventional
therapies\textsuperscript{1}. Among the heterogenous makeup of AML,
Monosomy7 (Mono7) is one of the most established poor prognostic markers
in AML with over four decades of clinical outcome data from a variety of
clinical trials and other studies\textsuperscript{2, 3, 4, 5}. Mono7 is
a rare event in pediatric AML resulting from the somatic copy number
loss of chromosome 7; despite intensive and myeloablative therapies,
outcomes for this subtype of AML remain dismal with no meaningful
advances in therapy.

We recently reported the discovery of fusions involving the Anaplastic
Tyrosine Kinase (\emph{ALK} ) gene and its unique occurrence in Mono7
pediatric AML\textsuperscript{6}. The \emph{ALK} gene encodes a receptor
tyrosine kinase, belonging to a family of protein kinases linked to
unregulated cell growth, that is a key component in CNS
development\textsuperscript{7}. \emph{ALK} is located on chromosome 2
(2p23) and chimeric ALK fusions have been frequently observed in cases
of inversion 2, as described in Non-Small Cell Lung Cancer (NSLC),
Anaplastic Large-Cell Lymphoma (ALCL), and
Neuroblastoma\textsuperscript{8, 9}. Additionally, \emph{ALK} is
integral to various molecular pathways involving proliferation and
differentiation in cells leading to a unique connection
between\emph{ALK} and tumorigenesis\textsuperscript{10}. The genetic
alterations described here involve Spectrin Beta Non-Erythrocytic 1
(\emph{SPTBN1} ), and RAN Binding Protein 2 (\emph{RANBP2} ), both of
which are located on chromosome 2. While previously reported in adult
AML and myelomonocytic leukemia, this report describes the first
instance of \emph{SPTBN1-ALK} and \emph{RANBP2-ALK} in pediatric
AML\textsuperscript{11, 12, 13}.

\emph{ALK} alterations in a variety of cancers have been observed to be
susceptible to targeted therapy with various kinase
inhibitors\textsuperscript{14, 15, 16}. \emph{ALK} positive
relapsed/refractory ALCL, Neuroblastoma, and other solid tumors in
children and young adults have been successfully treated with
crizotinib: an FDA-approved kinase inhibitor\textsuperscript{17, 18,
19}. Here we provide studies defining the functional consequence of
these ALK fusions (ALK\textsuperscript{fus}) demonstrating that these
fusions are transforming alterations and cells harboring these fusions
are sensitive to the ALK inhibitor crizotinib in a hematologic
environment. These data support the potential role of crizotinib to
therapeutically target this highly refractory cohort of Mono7 patients.

\emph{2 - Methods \& Results:}

\textbf{2.1 -- Fusion Cytogenetics}

As part of our recently completed discovery efforts in childhood AML
(TpAML), we interrogated the transcriptome of 1068 children and young
adults treated on the COG Phase 3 clinical trial, AAML1031, via RNA
sequencing. From these efforts, we identified cryptic and karyotypically
identified fusions involving the \emph{ALK} gene in 4 patients. The ALK
fusions included \emph{SPTBN1-ALK} (n=3) or \emph{RANBP2-ALK} (n=1)
(Fig. 1A)\textsuperscript{6}. Notably, all four
ALK\textsuperscript{fus}cases occurred exclusively in patients with
Mono7, constituting 14.3\% of patients with Mono7 (4 of 28); ALK fusions
were not detected in the remaining 1064 patients (Fig. 1B). In all four
cases the entire kinase domain of the \emph{ALK} gene was retained.
Importantly, overexpression of the \emph{ALK} gene was only identified
in the four ALK\textsuperscript{fus} positive cases with a mean TPM
expression of 11.76 versus 0 for ALK\textsuperscript{fus} negative
patients (Fig. 1B; p\textless{}0.0001), suggesting that
ALK\textsuperscript{fus}upregulates \emph{ALK} expression. The single
\emph{RANBP2-ALK} case additionally harbored chromosome inversion 2,
while only one of the\emph{SPTBN1-ALK} cases was noted to harbor
inversion 2; no other cytomolecular aberration was observed in any
ALK\textsuperscript{fus}cases (TABLE 1).

\textbf{2.2 -- Fusion Functionality}

To evaluate whether the ALK fusion transcripts generated oncoproteins,
fusion transcripts were cloned from patient samples into the pCSII
lentiviral vector (AddGene) under the EF1-Alpha promoter and tagged with
GFP, which was used to transduce and transfect cell lines to produce
model systems.

\emph{SPTBN1-ALK} or \emph{RANBP2-ALK} fusion transcripts were
transfected into HEK293T cells (ATCC), and cells were maintained in DMEM
supplemented with 10\% FBS, L-glutamine, and penicillin/streptomycin.
IL-3 dependent Ba/F3 cells were transduced with \emph{SPTBN1-ALK}
or\emph{RANBP2- ALK} fusion transcripts and maintained in RPMI 1640
supplemented with 10\% FBS, 10 ng/mL IL3, L- glutamine, and
penicillin/streptomycin. Cells were lysed with Triton X-Lysis buffer,
underwent western blotting (Invitrogen iBind), stained with anti-ALK
(Cell Signaling Technologies), and \selectlanguage{greek}β--\selectlanguage{english}Actin antibodies (Cell Signaling
Technologies). The presence of ALK protein was confirmed by western
blot, depicted as bands at approximately 124kD and 175kD (SPTBN1-ALK and
RANBP2-ALK, respectively) (Fig. 2A). The two protein species detected in
the RANBP2-ALK lysates suggest that the ALK region of the fusion may be
phosphorylated; this may suggest differential regulation of pathways
involving \emph{ALK} and would require further study.

The leukemic potential of the fusion transcripts were studied in a
transformation assay. Transduced Ba/F3 cells were sorted to GFP
homogeneity and growth kinetics post-IL-3 withdrawal were evaluated
using Trypan blue staining and live cell counting with Countess II
(Thermo Fischer Scientific).

Following IL-3 withdrawal from transduced Ba/F3 cells, cells expressing
either \emph{SPTBN1-ALK} or \emph{RANBP2-ALK} had sustained growth and
rapidly proliferated in cytokine-free media, whereas the parental line
quickly died in the absence of cytokines, confirming the transformation
potential of the fusion oncoprotein (Fig 2B). The presence of
the\emph{SPTBN1-ALK} led to greater cytokine-independent proliferation
compared to that of \emph{RANBP2-ALK} fusion.

\textbf{2.3 -- Cytotoxicity}

ALK inhibitors (e.g., crizotinib) are used in clinical practice for
various \emph{ALK} expressing malignancies therefore we hypothesized
that treatment with crizotinib would result in effective killing of
ALK\textsuperscript{fus} cells\textsuperscript{13}.
ALK\textsuperscript{fus}+ Ba/F3 cells were plated in triplicate and
cultured with varying doses of Crizotinib (SelleckChem) in cytokine-free
media\textsuperscript{20}. Cell viability and cell death were determined
with the Promega Cell Titer Glow assay after 48 hours in culture on a
BioTek Synergy H4 Hybrid plate reader via Gen5 2.1 Software; this was
repeated for ALK\textsuperscript{fus}+ cells in the presence of IL-3.
Crizotinib-induced cytotoxicity in ALK\textsuperscript{fus}+ Ba/F3 cells
was enhanced in the absence of IL-3 compared to cells cultured in the
presence of IL-3 (IC50 144nM vs. 1051nM for \emph{SPTBN1-ALK} and 95nM
vs. 1277nM for \emph{RANBP2-ALK} ) (Fig. 2C).

\emph{3 - Discussion:}

Identification of targetable lesions in AML provides clinicians with
novel treatment options to improve clinical outcomes for patients who
respond poorly to current conventional chemotherapy, such as patients
with Monosomy 7 AML. With ongoing discovery efforts and further
interrogation of large patient repositories, we hope to identify
additional novel targets, particularly for patients with rare subtypes
of AML.

Here, we demonstrate that newly discovered ALK\textsuperscript{fus} in
Mono7 AML leads to a functional ALK protein with the ability to
transform cells and induce a proliferative advantage in a
cytokine-independent manner. We show cells harboring the
ALK\textsuperscript{fus} to be susceptible to the ALK inhibitor
crizotinib, inducing tumor cell death in the context of AML \emph{in
vitro} modeling systems. We hypothesize treatment with newer generations
of ALK-inhibitors will result in more robust tumor cell killing and
warrants additional evaluation for exploration. Additionally, to
evaluate the preclinical efficacy of treating
ALK\textsuperscript{fus}AML with ALK-inhibitors, such as crizotinib,
future \emph{in vivo}experiments should be performed. The data presented
here provide rationale for ongoing exploration of crizotinib as an
effective therapeutic option for these high-risk patients with Monosomy7
AML.

\emph{Conflict of Interest:}

The authors have no relevant conflicts of interests to disclose.

\emph{Acknowledgements:}

We'd like to give special thanks to the St. Baldricks Foundation and the
Alex's Lemonade Stand Foundation for their funding of this project.
Additionally, we'd like to thank those enrolled in Children's Oncology
Group protocols and their families.

\emph{References:}

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\end{enumerate}\selectlanguage{english}
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