Cirmtuzumab (Zilovertamab) Eliminates ROR1 Positive Leukemia Stem Cells By Blocking Niche Dependent ROR1/WNT5a Signaling

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.

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.