Tumor microenvironment in CLL and MCL: pathogenesis, targets, and therapy

CLL 和 MCL 的肿瘤微环境：发病机制、靶点和治疗

• 批准号: 10706164
• 负责人: adrian u wiestner
• 金额: $ 166.39万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706164
• 关键词: Agammaglobulinaemia tyrosine kinase; B-Cell Activation; Binding; Biological; Bone Marrow; Cells; Cessation of life; Chronic; Chronic Lymphocytic Leukemia; Clinical; Clinical Data; Clinical Trials; Clonal Evolution; Conduct Clinical Trials; Consent; Data; Disease Progression; Dose; Down-Regulation; Enrollment; Epigenetic Process; Event; FDA approved; Gene Expression Profiling; Genetic Markers; Guanosine Triphosphate Phosphohydrolases; Immunity; Immuno-Chemotherapy; In complete remission; Inferior; Innovative Therapy; Intervention; Kinetics; Lactate Dehydrogenase; Mantle Cell Lymphoma; Mature B-Lymphocyte; Measurement; Measures; Minor; Modeling; Molecular; Multivariate Analysis; Mutagenesis; Mutation; NF-kappa B; Oncogenic; Outcome; PF4 Gene; PLCG2 gene; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Phase II Clinical Trials; Phenotype; Physiologic pulse; Pre-Clinical Model; Process; Progressive Disease; Proliferating; Publications; RNA; Refractory; Regimen; Relapse; Reporting; Resistance; Risk; Role; Safety; Sampling; Signal Transduction; Systems Biology; T cell factor 4; T-Lymphocyte; T-cell inflamed; TP53 gene; Testing; Therapeutic; Therapeutic Intervention; Time; Translations; Treatment Factor; Tumor-Derived Activated Cell; Tyrosine Kinase Inhibitor; Withdrawal; acquired drug resistance; activation-induced cytidine deaminase; base; chromosome 17p loss; chronic lymphocytic leukemia cell; cohort; design; disorder risk; exome sequencing; follow-up; genetic signature; high risk; high risk population; improved; in vitro Model; in vivo; inhibitor; large cell Diffuse non-Hodgkin' s lymphoma; lymph node microenvironment; lymph nodes; machine learning algorithm; macrophage; memory CD4 T lymphocyte; mouse model; novel strategies; patient derived xenograft model; patient stratification; patient subsets; peripheral blood; pre-clinical; prevent; prognostic model; prognostic value; randomized trial; relapse risk; response; risk stratification; single-cell RNA sequencing; small molecule inhibitor; targeted treatment; therapeutic target; therapy resistant; tool; transcriptome; treatment response; treatment risk; tumor; tumor microenvironment; Agammaglobulinaemia tyrosine kinase; B-Cell Activation; Binding; Biological; Bone Marrow; Cells; Cessation of life; Chronic; Chronic Lymphocytic Leukemia; Clinical; Clinical Data; Clinical Trials; Clonal Evolution; Conduct Clinical Trials; Consent; Data; Disease Progression; Dose; Down-Regulation; Enrollment; Epigenetic Process; Event; FDA approved; Gene Expression Profiling; Genetic Markers; Guanosine Triphosphate Phosphohydrolases; Immunity; Immuno-Chemotherapy; In complete remission; Inferior; Innovative Therapy; Intervention; Kinetics; Lactate Dehydrogenase; Mantle Cell Lymphoma; Mature B-Lymphocyte; Measurement; Measures; Minor; Modeling; Molecular; Multivariate Analysis; Mutagenesis; Mutation; NF-kappa B; Oncogenic; Outcome; PF4 Gene; PLCG2 gene; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Phase II Clinical Trials; Phenotype; Physiologic pulse; Pre-Clinical Model; Process; Progressive Disease; Proliferating; Publications; RNA; Refractory; Regimen; Relapse; Reporting; Resistance; Risk; Role; Safety; Sampling; Signal Transduction; Systems Biology; T cell factor 4; T-Lymphocyte; T-cell inflamed; TP53 gene; Testing; Therapeutic; Therapeutic Intervention; Time; Translations; Treatment Factor; Tumor-Derived Activated Cell; Tyrosine Kinase Inhibitor; Withdrawal; acquired drug resistance; activation-induced cytidine deaminase; base; chromosome 17p loss; chronic lymphocytic leukemia cell; cohort; design; disorder risk; exome sequencing; follow-up; genetic signature; high risk; high risk population; improved; in vitro Model; in vivo; inhibitor; large cell Diffuse non-Hodgkin' s lymphoma; lymph node microenvironment; lymph nodes; machine learning algorithm; macrophage; memory CD4 T lymphocyte; mouse model; novel strategies; patient derived xenograft model; patient stratification; patient subsets; peripheral blood; pre-clinical; prevent; prognostic model; prognostic value; randomized trial; relapse risk; response; risk stratification; single-cell RNA sequencing; small molecule inhibitor; targeted treatment; therapeutic target; therapy resistant; tool; transcriptome; treatment response; treatment risk; tumor; tumor microenvironment

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are tumors of mature B cells that are closely related biologically and currently incurable. BCR signaling has emerged as the pivotal pathway in the pathogenesis of both CLL and MCL. A major contribution from my group has been the first demonstration of active BCR signaling in CLL patients in vivo. Furthermore, we showed that BCR signaling and the consequent activation of the NF-kB pathway occurs primarily in the lymph node (LN) microenvironment rather than in the peripheral blood (PB) or bone marrow. Similarly, we found direct in vivo evidence for activation of the BCR and canonical NF-kB pathways in MCL that, in the absence of activating mutations, depends upon the LN microenvironment. These findings provide a mechanistic explanation for the surprising efficacy of Bruton Tyrosine Kinase inhibitors (BTKi) in treating MCL. To further characterize the intersection between microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired PB and LN samples from 34 patients. Oncogenic processes were upregulated in LN compared to PB. Single-cell RNA sequencing distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis revealed that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing of paired PB and LN samples showed subclonal expansion in LN in approximately half of patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution, but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives clinical outcome. Clonal trajectories are shaped by the LN milieu where T-cell immunity may contribute to suppress clonal outgrowth. Small molecule inhibitors of BCR signaling have transformed clinical approaches. The first-in-calls BTKi developed is ibrutinib. Between 2012 and 2014, our phase 2 clinical trial with ibrutinib enrolled 84 patients. Over twenty publications from my group report clinical and translational data from this particular study. We included 34 patients with TP53 alterations that were treated with single-agent ibrutinib. Historically, this groups of high-risk patients had a median survival of less than 3 years with intensive, multiagent chemoimmunotherapy. At a median follow-up of 6.5 years, 17 (50%) patients remained on study including six with complete responses. The most common reason for treatment discontinuation was disease progression (35%), followed by withdrawal of consents and deaths unrelated to CLL (6% each). Estimated 6-year progression-free (PFS) and overall survival was 61% and 79%, respectively. BTK and/or PLCG2 mutations were detected in 83% of progressors. We confirmed the long-term efficacy of ibrutinib in a pooled analysis of 89 patients with TP53 alterations. To improve tolerability and minimize off-target effects of ibrutinib, ACP-196 (acalabrutinib), a more BTK selective inhibitor was developed. Taking advantage of our PDX model, we contributed pre-clinical data supporting the translation of acalabrutinib into clinical trials. We investigated the safety, efficacy, and pharmacodynamics of acalabrutinib at 100 mg twice daily (BID) or 200 mg once daily (QD) in 48 patients with relapsed/refractory or high-risk treatment nave CLL. Acalabrutinib was well tolerated with over 90% of patients responding and an estimated PFS rate at 24 months of 79.2% and 91.5% with QD and BID dosing, respectively. Target occupancy, a measure of covalent binding of the drug to BTK, serves as a key pharmacodynamic parameter. The kinetics of de novo BTK synthesis, which determines durability of target occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remained undefined. Facilitated by a pulse-chase design of the first week of the study, we could estimate BTK resynthesis rate at 15% per day. BID dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared to QD dosing. Small increments in occupancy attained by BID dosing relative to QD dosing compounded over time to augment downstream biological effects. In conclusion, acalabrutinib was an effective and safe treatment for relapsed or high-risk treatment nave CLL patients. Twice daily dosing increased BTK occupancy and the consequent downregulation of oncogenic pathways. Measurements of target occupancy support the twice daily dosing regimen approved by the FDA. Randomized trials established the superiority of ibrutinib-based therapy over chemoimmunotherapy in patients with CLL. PFS is quite variable based on pre-treatment factors. To develop a risk stratification tool that can assist in the selection of therapeutic strategies, we analyzed 804 CLL patients treated with ibrutinib across six clinical trials and correlated the clinical model with genetic biomarkers of ibrutinib resistance. Multivariate analysis and machine learning algorithms identified four factors for a prognostic model that was validated in internal and external cohorts. Factors independently associated with inferior PFS and OS were: TP53 aberration, prior treatment, -2 microglobulin 5mg/L, and lactate dehydrogenase >250U/L. Each of these four factors contributed one point to a prognostic model that stratified patients into three risk groups: 3-4 points, high-risk; 2 points, intermediate-risk; 0-1 point, low-risk. The 3-year PFS for all 804 patients combined was 47%, 74% and 87% for the high-, the intermediate, and the low-risk group, respectively (P<0.0001). The 3-year OS was 63%, 83% and 93%, respectively (P<0.0001). In conclusion, the 4-factor model defines high-risk disease among CLL patients treated with ibrutinib. Following up on our studies with ibrutinib in patients with TP53 aberration we investigated the prognostic value of carrying isolated (single-hit) or multiple (multi-hit) TP53 aberrations. In our cohort, we identified TP53 mutations in 43 patients (84%) and del(17p) in 47 (92%); 9 and 42 patients carried single-hit and multi-hit TP53, respectively. PFS was significantly shorter in patients with multi-hit TP53 compared with those with single-hit TP53. Thus, single-hit TP53 defines a distinct subgroup of patients with an excellent long-term response to single-agent ibrutinib To identify mechanisms of BTKi resistance in CLL, in addition to BTK and PLCG2 mutations, we collaborated with external groups. In vitro modeling of resistance to ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. A phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in activating downstream NF-B. We confirmed a role for RAC2 also in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTKi. Clinically available drugs that can treat epigenetic ibrutinib resistance suggest possible therapeutic strategies.

慢性淋巴细胞性白血病（CLL）和地幔细胞淋巴瘤（MCL）是成熟B细胞的肿瘤，它们在生物学上密切相关且目前无法治愈。 BCR信号已成为CLL和MCL发病机理中的关键途径。我组的主要贡献是在体内CLL患者中首次证明了主动BCR信号传导。此外，我们表明BCR信号传导和随之而来的NF-KB途径的激活主要发生在淋巴结（LN）微环境中，而不是在外周血（PB）或骨髓中。同样，我们发现了MCL中BCR和规范NF-KB途径激活的直接体内证据，在没有激活突变的情况下，这取决于LN微环境。这些发现为Bruton酪氨酸激酶抑制剂（BTKI）在治疗MCL中的惊人功效提供了一种机械解释。为了进一步表征CLL发病机理中微环境与分子事件之间的交集，我们整合了来自34例患者的配对PB和LN样品的散装转录组分析。与PB相比，LN在LN中上调致癌过程。单细胞RNA测序区分了3个主要细胞状态：静止，激活和增殖。活化的亚群仅占LN样品总肿瘤总量的2.2％至4.3％。 RNA速度分析表明，LN中的Cll细胞命运是单向的，从增殖状态开始，过渡到活化状态，并以静止状态结束。源自活化肿瘤细胞的10基因签名与无治疗的生存率相关，并与LN中活化的CD4+记忆T细胞和M2巨噬细胞的比例正相关。对配对PB和LN样品的整个外显子组测序显示，大约一半的患者在LN中显示了亚克隆的扩张。由于小鼠模型在诱变中暗示了激活诱导的胞苷脱氨酶，因此我们比较了有或没有克隆进化的病例之间的AICDA表达，但没有发现差异。相反，LN中T细胞发炎的微环境的存在与克隆稳定性有关。总而言之，一个独特的小肿瘤亚群是CLL发病机理的基础，并驱动了临床结果。克隆轨迹由LN环境形成，在LN环境中，T细胞免疫力可能有助于抑制克隆的生长。 BCR信号传导的小分子抑制剂已转化了临床方法。开发的第一个呼叫btki是ibrutinib。 在2012年至2014年之间，我们与伊布鲁替尼进行的2期临床试验招募了84例患者。我小组的20多个出版物报告了这项特定研究的临床和翻译数据。 我们包括34例用单药ibrutinib治疗的TP53改变患者。从历史上看，这组高风险患者的中位生存期不到3年，并且具有强化，多基因的化学免疫性疗法。在6。5年的中位随访中，有17名（50％）的患者仍在研究中，其中6例具有完全反应。终止治疗的最常见原因是疾病进展（35％），然后撤回与CLL无关的同意和死亡（每个6％）。估计无6年的无进展（PFS）和总生存期分别为61％和79％。在83％的Progences中检测到BTK和/或PLCG2突变。我们在对89例TP53改变患者的合并分析中证实了依鲁替尼的长期疗效。 为了提高易夸替尼（ACP-196（Acalabrutinib））的耐受性并最大程度地减少靶向效果，开发了更多BTK选择性抑制剂。利用我们的PDX模型，我们贡献了临床前数据，该数据支持将acalabrutinib转化为临床试验。我们在48例复发/难治性或高危治疗中含Cll的患者中，每天两次（BID）两次（BID）或200 mg每天两次（BID）或200 mg每天两次或200毫克的Acalabrutinib的安全性，功效和药效学。阿卡拉略替尼的耐受性良好，有90％以上的患者和估计的PFS率分别为79.2％，分别为QD和BID剂量，为79.2％和91.5％。 目标占用率是该药物与BTK共价结合的量度，它是关键的药效参数。从头BTK合成的动力学决定了目标占用的耐用性以及占用，途径抑制和临床结果之间的关系仍然不确定。通过研究的第一周的脉冲练习设计，我们可以以每天15％的速度估计BTK重新合成率。 与QD剂量相比，竞标给药保持更高的BTK占用率，并获得了更有效的途径抑制。随着时间的推移，相对于QD剂量的出价剂量获得的占用率很小，以增强下游生物学效应。总之，阿卡劳替尼是对复发或高风险治疗中含CLL患者的有效且安全的治疗。每天两次给药增加了BTK的占用率，并导致致癌途径的下调。目标占用的测量支持FDA批准的两次每日给药方案。 随机试验确定了基于依鲁替尼在CLL患者的化学免疫疗法上的优越性。根据预处理因子，PFS是很大的。为了开发一种可以帮助选择治疗策略的风险分层工具，我们分析了804例在六项临床试验中用ibrutinib治疗的CLL患者，并将临床模型与依伯替尼耐药性的基因生物标志物相关联。多元分析和机器学习算法确定了在内部和外部队列中验证的预后模型的四个因素。与下pFS和OS独立相关的因素为：TP53像差，先前处理，-2微球蛋白5mg/L，乳酸脱氢酶> 250U/L。这四个因素中的每一个都为将患者分为三个风险组的预后模型贡献了一个点：3-4点，高风险； 2分，中等风险； 0-1点，低风险。所有804例患者合并的3年PF分别为47％，74％和87％，分别为中级和低风险组（P <0.0001）。 3年OS分别为63％，83％和93％（p <0.0001）。总之，四因素模型定义了用ibrutinib治疗的CLL患者中的高危疾病。 跟进我们对TP53畸变患者的ibrutinib的研究，我们研究了携带分离的（单次撞击）或多个（多重打击）TP53畸变的预后价值。在我们的队列中，我们确定了43例患者（84％）和47例DEL（17p）的TP53突变（92％）； 9和42例患者分别携带了单次击中和多击TP53。与单击TP53的患者相比，多点击TP53患者的PFS明显短。因此，单人击中TP53定义了对单药ibrutinib的长期反应的独特亚组 为了确定CLL中BTKI抗性的机制，除了BTK和PLCG2突变外，我们还与外部组合作。弥漫性大B细胞淋巴瘤激活的B细胞（ABC）亚型中对Ibrutinib的抗性的体外模型，该模型依赖于慢性活性BCR信号传导以生存。主要的抗性模式是表观遗传学的，部分由转录因子TCF4驱动。表型移动改变了BCR信号传导，使得GTPase RAC2在激活下游NF-B中代替BTK。我们证实了RAC2在BTKI上持续或进行性疾病患者的慢性淋巴细胞性白血病细胞中的作用。可以治疗表观遗传依鲁鲁替尼的临床可用药物可能会出现治疗策略。