ALK Genomic Aberrations in Head and Neck Squamous Cell Carcinoma (HNSCC)

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with high genetic heterogeneity, thus presenting a major challenge for precision medicine development. As of today, there are limited genomic events that are clinically actionable for HNSCC. Novel druggable genomic targets are urgently needed. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor with FDA-approved precision medicine for ALK-rearranged lung cancer. Other forms of ALK genetic aberrations are found in HNSCC, as well as in many other cancers. However, their biological roles and clinical implications are largely under-explored.Using next generation sequencing, we found ALK germline mutations in ~11% of Asian HNSCC patients in our in-house Asian HNSCC cohort (N=133). By and large, the therapeutic potential of these germline events is yet-to-be defined. Here, we first demonstrated that HNSCC patient-derived xenografts (PDXs) harboring a germline ALK p.W915fs mutation showed in vivo drug sensitivity to an FDA-approved ALK inhibitor, ceritinib. Subsequently, we showed that these PDXs were also sensitive to another very recently approved ALK inhibitor, lorlatinib. Whereas ALK-WT PDXs were not sensitive to ceritinib, nor lorlatinib. This novel finding suggests that ALK precision treatment may be effective for germline ALK-altered HNSCC. Further, HNSCC primary cultures with ALK germline mutations also conferred drug sensitivity to ceritinib, when compared to ALK-WT primary cultures.At the somatic level, analysis of the Cancer Genome Atlas (TCGA) HNSCC dataset (Firehose Legacy-HNSCC; N=510) revealed ~22% ALK genomic aberrations in HNSCC, including 3.6 % cases with ALK mutations, 0.4% and 18% cases with ALK amplification and copy number gain, respectively. Such a somatic mutation rate of ALK from TCGA HNSCC was validated in our in-house Asian HNSCC cohort (3%; 4/133 cases) as well. Strikingly, we found that ALK aberrations in HNSCC significantly co-occurred with PIK3CA aberrations (OR=2.40, P=0.045; TCGA Firehose Legacy-HNSCC, N=510). By functional genomics, we first reported that ALK amplification and seven HNSCC-associated ALK point mutations spanning intracellular kinase domain and extracellular domains were able to significantly drive HNSCC cell proliferation, clonogenicity, invasion and anoikis-resistance in PIK3CA-amplifed (AMP) HNSCC cells (FaDu), but not in PIK3CA-wildtype (WT) HNSCC cells (PECAPJ41). Importantly, PIK3CA mRNA knockdown by shRNA abrogated driver activities of ALK aberrations in PIK3CA-AMP HNSCC cells. Consistent with the in vitro driver activities noted, ALK aberrations promote the tumor growth in xenografts of PIK3CA-AMP HNSCC cells, while PIK3CA shRNA knockdown attenuated such in vivo tumor growth. Our findings indicate that ALK aberrations are corroborating with PIK3CA aberrations in HNSCC for tumorigenesis. Consistent with previous reports with ALK-rearranged lung cancer cells, our RNA-seq transcriptional profiling showed the downregulated signature of mRNA metabolism and catabolism. RNA-seq analysis of pathway signatures revealed that different HNSCC-associated ALK mutations could activate different major signaling pathways to exhibit their driver activities in PIK3CA-AMP HNSCC.Over 80% of HNSCC patients with co-occurring ALK/PIK3CA aberrations have advanced diseases (Stage III, or IV) (TCGA, Firehose Legacy-HNSCC). Using HNSCC patient-derived primary cultures with an endogenous PIK3CA mutation, we identified for the first time the natural emergence of a somatic ALK mutation during anoikis-resistance progression, supportive of the clinical observation of co-existence of ALK/PIK3CA aberrations in HNSCC patients, likely linked to aggressive progression of HNSCC. HNSCC with such an emerged ALK mutation showed enhanced cell proliferation and invasion, and most importantly heightened sensitivity to ALK inhibition by ceritinib in vitro and in vivo. Most importantly, we identified that several HNSCC-associated somatic ALK mutations could confer heightened drug sensitivity to ceritinib in vitro and in vivo. Moreover, combination therapy of ALK and PI3K inhibitors demonstrated marked therapeutic efficacy in ALK/PIK3CA-altered HNSCC PDXs and xenografts. In conclusion, HNSCC-associated ALK aberrations are functional drivers in corroboration with PIK3CA aberrations in HNSCC. Targeting of both ALK germline and somatic mutations with ALK inhibitors has potential clinical benefits for HNSCC patients, which warrants clinical investigations in the future.頭頸部鱗狀細胞癌（HNSCC）是一種具有高度遺傳異質性的侵襲性癌症，因此對精準醫學的發展提出了重大挑戰。截至今天，HNSCC在臨床上可應用的基因組事件有限, 迫切需要新的可藥物治療的基因組靶標。間變性淋巴瘤激酶（ALK）是酪氨酸激酶受體， 臨床上已有FDA批准用於ALK變異肺癌的靶向藥物。在HNSCC以及許多其他癌症中也發現了其他形式的ALK基因畸變。然而，它們的生物學作用和臨床意義在很大程度上尚未得到開發。通過下一代基因測序，我們分析了亞洲HNSCC隊列（N = 133）並發現了約11％的亞洲HNSCC患者具有ALK種系突變，然而這些種系事件的治療潛力尚待確定。在這裡，我們首先證明了帶有種系ALK p.W915fs突變的HNSCC患者來源腫瘤異種移植（PDX）對FDA批准的ALK抑製劑ceritinib表現出有效的體內藥物反應。隨後，我們表明該PDX對另一種最近批准的ALK抑製劑lorlatinib也表現有效的藥物反應。而ALK-WT PDX對ceritinib或lorlatinib均沒有藥物反應。這一新發現表明 ALK 精確治療可能對種系ALK突變的 HNSCC 有效。此外，與ALK-WT原代培養細胞相比，具有ALK種系突變的HNSCC原代培養細胞也對ceritinib有藥物反應。在體細胞水平上，從對美國癌症基因體圖譜計畫（TCGA）的HNSCC數據庫（Firehose Legacy-HNSCC； N = 510）的分析顯示，HNSCC中約有22％的ALK基因組畸變，包括3.6％的ALK突變，0.4％擴增和18％拷貝數增益。 TCGA HNSCC中的ALK種體細胞突變率也在亞洲HNSCC隊列中也得到了驗證（3％； 4/133例）。值得注意的是，我們發現在HNSCC 中的ALK 畸變與 PIK3CA 畸變顯著共同發生（OR=2.40，P=0.045；TCGA Firehose Legacy-HNSCC，N=510）。通過功能性研究，我們首次報導了ALK擴增和HNSCC相關的七個與跨越細胞內激酶結構域和細胞外結構域的ALK點突變能夠顯著驅動有PIK3CA擴增的（AMP）HNSCC細胞 (FaDu)的細胞增殖，克隆形成，侵襲和抗凋亡。但在 PIK3CA 野生型 (WT) HNSCC 細胞 (PECAPJ41) 中並沒有發現ALK畸變的驅動活性。值得注意的是shRNA敲低PIK3CA mRNA可以阻止PIK3CA-AMP HNSCC細胞中ALK畸變的驅動活性。與體外驅動活性一致，ALK畸變也促進了PIK3CA-AMP HNSCC細胞異種移植中體內腫瘤的生長，然而PIK3CA shRNA敲低卻減弱了這種體內腫瘤的生長。我們的發現證實了PIK3CA畸變與ALK畸變在HNSCC中共同合作以促進腫瘤生長。與先前關於ALK陽性的肺癌細胞的研究一致，我們的RNA-seq數據分析顯示了mRNA代謝和分解代謝的轉錄譜的變化。通路特徵的RNA序列分析表明，不同的HNSCC相關的ALK突變可以激活不同的主要信號通路，以顯示其在PIK3CA-AMP HNSCC中的驅動活性。在ALK / PIK3CA畸變共存的HNSCC患者中，超過80％的患者患有晚期疾病（III期或IV期）（TCGA，Firehose Legacy-HNSCC）。在具有內源性PIK3CA突變的HNSCC患者原代培養細胞的抗失巢凋亡篩選過程中，我們首次發現了體細胞ALK突變的自然出現，這一發現支持了臨床上所觀察到的ALK/PIK3CA 畸變共存的HNSCC 患者，可能與 HNSCC 的侵襲性進展有關。具有這種出現的ALK突變的HNSCC細胞表現出更強的細胞增殖和侵襲的能力，並且提高了在體外和體內對ALK抑制劑ceritinib的有效藥物反應。最重要的是，我們確定了HNSCC相關的體細胞ALK突變可以在體外和體內對ceritinib表現出更好的有效藥物反應。此外，ALK和PI3K抑製劑的聯合治療在ALK / PIK3CA畸變的HNSCC PDX和異種移植中顯示出顯著的治療效果。總括而言，在HNSCC中ALK畸變與PIK3CA畸變互相合作以驅動侵襲性腫瘤生長。用ALK抑製劑靶向ALK種系和體細胞突變對HNSCC具有潛在的臨床益處，這值得在將來進行進一步的臨床研究。WANG, Lan.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...