Ang Zhiwei, Paruzzo Luca, Hayer Katharina E, Schmidt Carolin, Torres Diz Manuel, Xu Feng, Zankharia Urvi, Zhang Yunlin, Soldan Samantha, Zheng Sisi, Falkenstein Catherine D, Loftus Joseph P, Yang Scarlett Y, Asnani Mukta, King Sainos Patricia, Pillai Vinodh, Chong Emeline, Li Marilyn M, Tasian Sarah K, Barash Yoseph, Lieberman Paul M, Ruella Marco, Schuster Stephen J, Thomas-Tikhonenko Andrei
bioRxiv. 2023 Aug 16:2023.02.19.529123. doi: 10.1101/2023.02.19.529123.
Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the gene encoding human CD20 also produces several mRNA isoforms with distinct 5' untranslated regions (5'-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.
In normal & malignant human B cells, CD20 mRNA is alternatively spliced into four 5'-UTR isoforms, some of which are translation-deficient.The balance between translation-deficient and -competent isoforms modulates CD20 protein levels & responses to CD20-directed immunotherapies.
We discovered that in normal and malignant B-cells, CD20 mRNA is alternatively spliced to generate four distinct 5'-UTRs, including the longer translation-deficient V1 variant. Cells predominantly expressing V1 were still sensitive to CD20-targeting chimeric antigen receptor T-cells. However, they were resistant to the bispecific anti-CD3/CD20 antibody mosunetuzumab, and the shift to V1 were observed in CD20-negative post-mosunetuzumab relapses of follicular lymphoma.
CD19和CD22中编码外显子的异常跳跃会影响B细胞恶性肿瘤免疫治疗的效果。在此，我们发现编码人类CD20的基因也会产生几种具有不同5'非翻译区（5'-UTR）的mRNA亚型。通过RNA测序在正常B细胞分化和B淋巴细胞恶性肿瘤的不同阶段可检测到四种变体（V1-4），其中V1和V3最为丰富。在B细胞激活和爱泼斯坦-巴尔病毒感染期间，剪接从V1重定向到V3与CD20阳性增加同时发生。同样，在弥漫性大B细胞淋巴瘤中，只有V3而不是V1与CD20蛋白水平相关，这表明V1可能缺乏翻译能力。事实上，发现较长的V1亚型包含上游开放阅读框（uORF）和茎环结构，它们共同抑制多核糖体募集。通过用剪接转换吗啉代寡聚物调节CD20亚型，我们增强了一组B细胞系中CD20的表达以及抗CD20抗体利妥昔单抗介导的细胞毒性。此外，用V3 mRNA重建CD20基因敲除细胞导致CD20阳性恢复，而用V1重建的细胞中CD20蛋白水平检测不到。令人惊讶的是，靶向CD20的嵌合抗原受体T细胞能够杀死表达V3和V1的细胞，但双特异性T细胞衔接分子莫苏奈妥珠单抗仅对表达V3的细胞有效。为了确定CD20剪接是否参与免疫治疗抗性，我们对4例莫苏奈妥珠单抗治疗后复发的滤泡性淋巴瘤进行了RNA测序，发现其中2例CD20的下调伴随着V3到V1的转变。因此，剪接介导的表位丢失机制扩展到了靶向CD20的免疫治疗。
在正常和恶性人类B细胞中，CD20 mRNA可选择性剪接成四种5'-UTR亚型，其中一些缺乏翻译能力。缺乏翻译能力和具有翻译能力的亚型之间的平衡调节CD20蛋白水平以及对靶向CD20的免疫治疗的反应。
我们发现，在正常和恶性B细胞中，CD20 mRNA可选择性剪接以产生四种不同的5'-UTR，包括较长的缺乏翻译能力的V1变体。主要表达V1的细胞对靶向CD20的嵌合抗原受体T细胞仍然敏感。然而，它们对双特异性抗CD3/CD20抗体莫苏奈妥珠单抗具有抗性，并且在莫苏奈妥珠单抗治疗后复发的滤泡性淋巴瘤CD20阴性病例中观察到向V1的转变。
