5-Azacytidine prevents relapse and produces long-term complete remissions in leukemia xenografts treated with Moxetumomab pasudotox

Abstract

Moxetumomab pasudotox (Moxe) is a chimeric protein composed of an anti-CD22 Fv fused to a portion of exotoxin A and kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia, but many children with relapsed/refractory acute lymphoblastic leukemia (ALL) either respond transiently or are initially resistant. Resistance to Moxe in cultured cells is due to low expression of diphthamide genes (DPH), but only two of six ALL blast samples from resistant patients had low DPH expression. To develop a more clinically relevant model of resistance, we treated NSG mice bearing KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells were killed by Moxe, but relapse occurred from discrete bone marrow sites. The resistant cells would no longer grow in cell culture and showed major chromosomal changes and changes in phenotype with greatly decreased CD22. RNA deep sequencing of resistant KOPN-8 blasts revealed global changes in gene expression, indicating dedifferentiation toward less-mature B cell precursors, and showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival increased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells and is prevented by treatment with 5-azacytidine.

Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy

“…However, reduced diphthamide synthesis gene expression was also observed in immunotoxin-responsive patient cells. It was concluded that diphthamide gene expression could not account for the majority of resistant cases [ 54 ]. Mouse models injected with GFP/luciferase-expressing KOPN-8 or Reh cells revealed that a few resistant cells persisted in the bone marrow and subsequently expanded systemically.…”

“…Significant chromosomal alterations were also observed. Interestingly, 5-azacytidine prevented this resistance in one mouse model and delayed it in another, though it was unable to overcome established resistance [ 54 ].…”

A rare subgroup of leukemia stem cells harbors relapse-inducing potential in acute lymphoblastic leukemia

“…The fact that dormancy and resistance are reversible upon release from the microenvironment indicates that transcriptional reprogramming of ALL-LIC is an early event in the generation of therapy resistance. This process may precede the onset of hardwired irreversible resistance and relapse, and a similar multistep-mechanism of the emergence of resistant disease has recently been suggested under treatment with the immunotoxin Moxetumomab pseudotox [109]. The in vivo model described in our recent study [68], in combination with CRISPR-Cas9 edited NGS mice [91] can facilitate dissecting regulatory factors within the bone marrow microenvironment that endow LIC with adverse characteristics.…”

DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance

“…Therefore, it is possible that resistance mechanisms may be shared across this class of toxin immunoconjugates. However, while in vitro resistance to moxetumomab pasudotox in cell lines was associated with downregulation of diphthamide synthesis pathway genes (21), this did not seem to be the dominant mechanism of resistance in B-ALL treated in vivo (30). Rather, moxetumomab resistance was associated with a developmental state change, karyotypic abnormalities, and loss of surface CD22 expression.…”

“…Rather, moxetumomab resistance was associated with a developmental state change, karyotypic abnormalities, and loss of surface CD22 expression. Yet, despite this difference, azacitidine and moxetumomab were also an effective combination in B-ALL cell lines treated in vivo (30). Thus, mechanisms of in vivo resistance to diphthamide-targeting toxins may be disease or celltype dependent.…”