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 Pseudomonas 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.

Trial registration: ClinicalTrials.gov NCT01891981.

Keywords: 5-azacytidine; CD22; Moxetumomab pasudotox; acute lymphoblastic leukemia; immunotoxin.

Conflict of interest statement

Conflict of interest statement: A.S.W. and I.P. are coinventors on patents assigned to the NIH for the investigational products. A.S.W. has received research support, honorarium, and travel support from Medimmune; and honorarium and travel support from Pfizer, Kite Pharma, and Spectrum Pharmaceuticals.

Figures

Fig. 1.

Decreased DPH-gene expression correlates with resistance of one-third of the tested ALL samples. The reads for the indicated DPH enzymes were quantified by RNA deep sequencing of a total of seven Moxe-sensitive cell lines, normalized to reads per kilobase and million mapped reads, averaged, and set to 1. (A) The reads of the DPH-enzymes of six Moxe-resistant and two Moxe-sensitive patients were similarly determined by RNA deep sequencing and then normalized to the average of the seven sensitive cell lines. Nonresponder 6 was determined in duplicates all other patient reads were measured only once. (B) To determine the relative expression levels of the previously published resistant cell lines (16, 17), the DPH-expression was determined for their respective Moxe-sensitive cell line and multiplied by the changes in DPH for the resistant subclones, as determined by RT-PCR. The RT-PCR changes were determined in triplicates (16, 17).

Fig. 2.

Moxe treatment clears the BM from ALL but disease relapses from discrete BM-sites. (A) Kaplan–Meyer survival curve for mice treated with three doses of 0.4 mg/kg Moxe every other day from day 8 or vehicle. The P value was determined by log-rank test. (B) Rate of BM infiltration for KOPN-8 bearing mice treated with the schedule in A and killed at indicated times. Symbols show individual mice, lines indicate means, error as SD, significance determined by unpaired t test, with P value as ns = not significant, ***P < 0.001. (C) Mice were injected with KOPN-8L and treated with three doses of 0.4 mg/kg Moxe every other day or vehicle from day 8. Moxe-treatment was repeated from day 29 (red arrows). Bioluminescence signal was determined at treatment start (day 8) and at indicated days. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 100–30,000. (D–F) Mouse 2 was killed, the right leg excised, formalin-fixed, paraffin-embedded, and stained for a human-specific mitochondrial marker (Mito-A/B, peroxidase signal). Images were digitalized using 40-fold optical magnification.

Fig. 3.

Leukemia relapse stays sensitive to retreatment for several cycles. Mice bearing KOPN-8L cells were treated with two (A), or three (B) cycles of Moxe from days 8, 38, and 57, as indicated by the arrows. One cycle consisted of three doses of 0.4 mg/kg Moxe every other day. Bioluminescence was measured at indicated days after leukemia inoculation. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 100–30,000. Shown are representative images of four independent experiments with similar results. (C) RehL-bearing mice were treated with three cycles of Moxe from days 6, 27, and 41. Mice were imaged at indicated days. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 200–30,000 with the exception of days 48 and 59, which were scaled from a radiance of 1,000–30,000 to reduce background.

Fig. 4.

Moxe-resistant relapse shows major phenotype and chromosome changes. KOPN-8L and KOPN-8L-R were engrafted in five mice each, their BM sorted for ALL cells, which were split to produce total RNA and total protein. (A) mRNA levels by real-time PCR in KOPN-8L-R from BM or control KOPN-8-R from cell culture (CC) (16) relative to KOPN-8L (dotted line). Bars indicate average fold-changes of CT-values in triplicates, error as SD. P values as ns = not significant, *P < 0.033, **P < 0.002, ***P < 0.001, ****P < 0.0001. (B) The BM-derived ALL blasts from five independent mice bearing KOPN-8L or KOPN-8L-R were measured for the indicated markers by flow cytometry, t tests determined P values as ns = not significant, *P < 0.033, **P < 0.002, ***P < 0.001. (C) Western blot for total CD22 protein of BM-derived ALL blasts from five independent mice, densitometry values are actin normalized. (D) BM from three individual mice bearing RehL or RehL-R (from Fig. 3C) were analyzed as described in B. *P < 0.033, **P < 0.002, ***P < 0.001. (E) KOPN-8 GL (GL) cells were cultured alone or GL cells and GL-R cells were cultured with OP-9 stromal cells, treated with Moxe, viability determined by flow cytometry for the GFP+ cells, which was normalized to untreated control (100%) and 0% is a true 0. Each symbol represents the mean percentage of living cells in triplicates, error as SD. (F) Chromosome analysis by spectral karyotyping showing the karyotype of KOPN-8L [44,XX, add(8q), t(11;19), t(13;14), −13, −14] acquired six additional chromosomal aberrations in KOPN-8L-R [+6, +7, +8, +t(8;5) ×2, and +20]; >10 karyotypes were analyzed, shown is the representative. (G) The expression level of the fusion transcript of the KMT2A (chromosome 11) and the MLLT1 (chromosome 19) translocation was measured by RNA deep sequencing, as described in Materials and Methods. Each symbol represents the number of uniquely mapped reads of individual mice, bars represent the average, error as SD, P values were determined by unpaired t test.

Fig. 5.

RNA-deep sequencing confirms profound changes in KOPN-8L-R. BM-derived cells were FACS-sorted and their RNA isolated for RNA-deep sequencing. (A) The mapped reads were counted, normalized to counts per million (cpm), log2-transformed, and each individual gene from resistant cells (Res, x-coordinate) plotted against sensitive cells (Sens, y-coordinate). Every dot represents the average log2 (cpm) from five independent mice. Highlighted are B cell marker genes (blue rhombi) that are down-regulated and non-B cell marker genes (red stars) that are up-regulated in KOPN-8L-Res. (B) All genes were preranked using limma’s t-statistic from most up-regulated to most down-regulated and the list was compared with MSigDb gene lists. The total number of genes and the number of overlapping genes between a pathway and the experimentally determined gene list are indicated. Every gene present in the preranked gene list and in the respective pathway is represented by a black line above the red (up-regulated) to blue bar (down-regulated), indicating the gene’s rank in the preranked list. The enrichment score (ES) (y axis) is generated by an algorithm, which increases a “running-sum statistic” when a gene is present in the preranked list or decreases when the gene is absent [green line (23)]. The enrichment score is then normalized to the gene set size, resulting in the NES (23). The NES and the false-discovery rate “q” are indicated. DN, down-regulation; PID, pathway interaction database; UP, up-regulation. (C) Shown are TFs central to the early development of B cells, common lymphoid progenitor (CLP), lymphoid-primed multipotent progenitor (LMPP), multipotent progenitor (MPP), and prepro-B cells. Indicated by dashed arrows are interactions between the TF driving development toward B cells or DCs. The fold up- (blue) and down-regulation (green) of TFs in KOPN-8L-Res are shown. Graph adapted from ref. .

Fig. 6.

The combination of 5-AZA and Moxe greatly improves responses of the KOPN-8L xenografts. Mice inoculated with KOPN-8L were treated with vehicle, five doses of 0.3 mg/kg Moxe every other day from day 8, 2 mg/kg 5-AZA from day 3, or the combination. Bioluminescence was determined before treatment start on day 8 and at indicated days. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 100–30,000. Mice shown in A and B were inoculated with KOPN-8L and mice shown in C. with KOPN-8L-R. Symbols in B and C, Upper represent the average bioluminescence intensities per mouse in the respective treatment group; P values were determined by t test. The respective Kaplan–Meyer survival graphs are shown in the in B, Lower, for KOPN-8L mice and C for KOPN-8L-R; P values were determined by log-rank test. t test-derived P values as ns = not significant, *P < 0.033, **P < 0.01, ****P < 0.0001.

Fig. 7.

5-AZA enhances Moxe activity in the RehL xenograft. (A) Mice were inoculated with RehL cells expressing GFP/luciferase and treated with vehicle, five doses of 0.3 mg/kg Moxe every other day from day 8, 2 mg/kg 5-AZA intraperitoneally from day 3, or the combination. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 100–30,000. (B) Kaplan–Meyer survival graph for mice in A; P values were determined by log-rank test as ns = not significant, ***P < 0.001, ****P < 0.0001.