Abstract
Background: While not expressed in healthy adult tissues, ROR1 is an embryonic tyrosine kinase-like molecule with high
level expression in CLL, ALL and many solid tumors. Expression of ROR1 makes these malignancies ideal
targets for therapy with Zilovertamab (cirmtuzumab), a highly selective anti-human ROR1 monoclonal
antibody that is currently in clinical trials for the treatment of CLL and other malignancies. Meanwhile, very
little is known about ROR1 expression in AML, a highly lethal disease with only 20% of patients surviving
for 5 years. In AML, therapeutic resistance is driven by CD34+ leukemia stem cells (LSC) harboring
enhanced survival, dormancy and self-renewal capacity in supportive niches. Deregulation of an innate
immune adenosine to inosine (A-to-I) RNA deaminase (ADAR1) augments LSC propagation in response
to inflammatory cytokine induced JAK2/STAT signaling. Thus, we investigated a possible link between
ROR1 and ADAR1 activation in LSC maintenance and Cirmtuzumab (now known as Zilovertamab) as a
potential LSC eradication strategy.
Results: First, we focused investigated expression of CD34+ and ROR1+ on subpopulations of AML cells by FACS,
whole transcriptome RNA sequencing (RNA-seq) and single cell proteogenomics (Tapestri®) analyses. Our
data demonstrated that about 42% of primary adult AML (de novo and sAML) and 50% of pediatric AML
samples co-expressed ROR1 and CD34. Zilovertamab induced dose-dependent inhibition of survival and
replating in ROR1+ samples with no cytotoxicity toward normal CD34+ cells. In contrast to clonogenic
survival assays, where the response of AML HPC to Zilovertamab was 100%, replating (self-renewal)
assays demonstrated that 67% of AML LSC were responsive and 33% were non-responsive. Notably, all
ROR1+ pediatric AML samples were responsive and had significantly higher LSC inhibition in response to
Zilovertamab compared to adult de novo AML but similar to sAML. There was a negative correlation
between ADARp150 and response of the samples to Zilovertamab treatment. To investigate clonal DNA
mutational and phenotypic hierarches in AML, we performed multiplexed single cell proteogenomics using
a mixture of antibody-oligo conjugates, CD3, CD7, CD11b, CD34, CD38, CD45, CD56, and ROR1, with
simultaneous analysis of DNA mutations in 45 myeloid malignancy-associated genes covering 312
amplicons. Immunophenotyping showed ROR1 positive single cells harbored mutations that promote
therapeutic resistance, such as mutations in TP53. To understand the downstream signaling of ROR1 and
interaction with its ligands, three pairs of AML cell lines were transduced with ROR1 and co-cultured with
stroma producing human cytokines or recombinant WNT5a and were treated with Zilovertamab or aWNT5a mAb.
Interaction of AML cells with SLM2 stroma, which has enhanced IL3 production, resulted in
the highest level of proliferation. ROR1 expression boosted AML cell clonogenic potential, particularly in
the presence of stroma. In ROR1+ cells, Zilovertamab inhibited colony formation in a dose dependent
manner. In contrast to controls, in ROR1+ AML cells clonogenic capacity could be blocked by both
Cirmtuzumab (Zilovertamab) and WNT5a inhibitory Ab, suggesting the specificity of ROR1 and WNT5a
interactions.
Conclusions: This study demonstrates that an anti-ROR1 mAb, Zilovertamab, can eliminate up to 80% of AML LSC in
supportive niches. In the future, targeted ROR1 inhibition may represent a vital component of therapeutic
strategies aimed at eradicating therapeutically recalcitrant LSCs in AML and potentially other refractory
cancer stem cell-driven malignancies.
Disclosures
Kipps:Gilead: Consultancy; Pharmacyclics, LLC an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Consultancy; Genentech-Roch: Consultancy, Research Funding; Oncternal: Research Funding. Jamieson:Aspera Biomedicines, Impact Biomedicines,: Consultancy, Other: co-founder of Aspera Biomedicines and Impact Biomedicines and has received royalties for intellectual property licensed by Forty Seven Inc, Patents & Royalties: Forty Seven Inc.
Author notes
Asterisk with author names denotes non-ASH members.
