Secondary Lesions and Sensitivity to Signaling Inhibitors of Pediatric iAMP21 B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction

Intrachromosomal amplification of chromosome 21 (iAMP21) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children is a high-risk subtype for which targeted drugs are lacking. In this study we aimed to determine the frequency of secondary lesions and investigated the cellular sensitivity for candidate targeted drugs.

Methods

We performed total RNA sequencing on 28 iAMP21 and 28 B-other (negative for sentinel fusion genes) pediatric samples to determine the frequency of secondary lesions in newly diagnosed patients. A panel of 18 patient derived xenografts (PDX) of 8 primary iAMP21 ALL samples was generated, and secondary lesions were validated by PCR, RT-PCR, and whole exome sequencing. To test sensitivity, primary or PDX cells were exposed ex vivo to a concentration range of gilteritinib (FLT3 inhibitor), trametinib (MEK1/2 inhibitor), or ruxolitinib (JAK1/2 inhibitor).

Results

Secondary lesions in the cytokine receptor gene FLT3 were enriched in iAMP21 compared with B-other ALL including internal tandem duplications (ITD) and other activating lesions (50.0% vs. 10.7%, p=0.003). Lesions in genes encoding cytokine receptors CRLF2 and IL7R had a similar frequency between iAMP21 and B-other cases (25% vs. 17.9%, p=0.75 and 7.1% vs. 0%, p=0.49 respectively). Inactivating lesions in SH2B3, the downstream negative regulator of JAK/STAT and FLT3 signalling, were more frequent in iAMP21 cases vs. B-other (46.4% vs. 7.1%, p=0.002), while the frequency of JAK1 and JAK2 mutations did not differ (3.6 vs. 0% and 10.7 vs. 10.7% p = 1 for both). All SH2B3, CRLF2 and JAK lesions were retained in PDX samples, whereas in contrast FLT3-ITD was retained in only 2 of 5 PDX.

Gilteritinib sensitivity did not differ between iAMP21 and B-other cases (median LC50 1.24 µM, range 0.33 to 4.85 µM vs. median LC50 1.21 µM, range 1.20 to 1.3 µM, p=0.95). Grouping iAMP21 cases by FLT3 and SH2B3 status, samples with both FLT3-ITD and SH2B3 lesion had the highest sensitivity to gilteritinib (median LC50 0.39 µM, range 0.35 to 0.43 µM). Samples with only FLT3 or SH2B3 lesion did not show increased sensitivity compared to those without a lesion (p>0.5 for both). Median ruxolitinib sensitivity did not statistically differ between iAMP21 and B-other cases (median LC50 8.38 µM, range 1.00 to >10 µM, vs. median LC50 0.48, range 0.21 to >10 µM; p=1) although extreme resistance to ruxolitinib seemed more frequent in iAMP21 cases (6 out of 12 cases) and was not related to FLT3 or SH2B3 status. CRLF2-rearranged ( CRLF2r) iAMP21 cases were slightly more sensitive to ruxolitinib than those lacking CRLF2r, although this difference did not reach significance (median LC50 4.72 µM, range 1.0-4.85 vs. median LC50 >10 µM, range 2.66 to >10 µM; p=0.08). The highest sensitivity was found in the only case with both a CRLF2r and a JAK1 mutation (LC50 of 1.00 µM). A large variation was present in trametinib sensitivity amongst both iAMP21 and B-other cases (median LC50 0.11 µM, range 0.017 to >5 µM vs. median LC50 0.18, range 0.018 to >5 µM; p=0.72). More than half of iAMP21 cases were sensitive irrespective of RAS-pathway lesion status.

Conclusion

iAMP21 leukemias are enriched in FLT3 and in SH2B3 lesions which, when co-occurring, affect sensitivity to FLT3 inhibition by gilteritinib but do not affect JAK-inhibition by ruxolitinib. This suggests these lesions act synergistically and might bypass downstream JAK/STAT signalling. This might also explain the observed sensitivity to RAS-pathway inhibition irrespective of secondary lesions. These results suggest that further research into FLT3 and RAS signalling inhibitors might lead to better treatment options for pediatric iAMP21 BCP-ALL.